Intracellular Replication-Deficient Leishmania donovani Induces Long Lasting Protective Immunity against Visceral Leishmaniasis This information is current as of September 26, 2021. Angamuthu Selvapandiyan, Ranadhir Dey, Susanne Nylen, Robert Duncan, David Sacks and Hira L. Nakhasi J Immunol 2009; 183:1813-1820; Prepublished online 10 July 2009; doi: 10.4049/jimmunol.0900276 Downloaded from http://www.jimmunol.org/content/183/3/1813

Supplementary http://www.jimmunol.org/content/suppl/2009/07/13/jimmunol.090027 http://www.jimmunol.org/ Material 6.DC1 References This article cites 42 articles, 25 of which you can access for free at: http://www.jimmunol.org/content/183/3/1813.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Intracellular Replication-Deficient Leishmania donovani Induces Long Lasting Protective Immunity against Visceral Leishmaniasis

Angamuthu Selvapandiyan,* Ranadhir Dey,* Susanne Nylen,† Robert Duncan,* David Sacks,† and Hira L. Nakhasi1*

No vaccine is currently available for visceral leishmaniasis (VL) caused by Leishmania donovani. This study addresses whether a .live attenuated centrin gene-deleted L. donovani (LdCen1؊/؊) parasite can persist and be both safe and protective in animals LdCen1؊/؊ has a defect in amastigote replication both in vitro and ex vivo in human macrophages. Safety was shown by the lack of parasites in spleen and liver in susceptible BALB/c mice, immune compromised SCID mice, and human VL model hamsters 10 ؊/؊ wk after infection. Mice immunized with LdCen1 showed early clearance of virulent parasite challenge not seen in mice Downloaded from immunized with heat killed parasites. Upon virulent challenge, the immunized mice displayed in the CD4؉ T cell population a significant increase of single and multiple cytokine (IFN-␥, IL-2, and TNF) producing cells and IFN-␥/IL10 ratio. Immunized mice also showed increased IgG2a immunoglobulins and NO production in macrophages. These features indicated a protective Th1- type immune response. The Th1 response correlated with a significantly reduced parasite burden in the spleen and no parasites in the liver compared with naive mice 10 wk post challenge. Protection was observed, when challenged even after 16 wk post immunization, signifying a sustained immunity. Protection by immunization with attenuated parasites was also seen in hamsters. http://www.jimmunol.org/ -Immunization with LdCen1؊/؊ also cross-protected mice against infection with L. braziliensis that causes mucocutaneous leish maniasis. Results indicate that LdCen1؊/؊ can be a safe and effective vaccine candidate against VL as well as mucocutaneous leishmaniasis causing parasites. The Journal of Immunology, 2009, 183: 1813–1820.

he obligate kinetoplastid protozoan parasites of the genus So far, several procedures have been used to develop live at- Leishmania are spread by sand fly vectors and cause a tenuated Leishmania parasites including long-term in vitro cul- spectrum of diseases collectively known as leishmaniasis tures, selection for temperature sensitivity, chemical mutagenesis, T 2 that ranges from self-healing cutaneous leishmaniasis (CL), mu- and irradiation (1, 2). Although such live attenuated lines have by guest on September 26, 2021 cocutaneous leishmaniasis (MCL), and to fatal visceral leishman- shown substantial protection against challenge in animal models, iasis (VL). Leishmaniasis is endemic to 88 countries in the tropical undefined random genetic mutations and concerns arising from and subtropical world, affecting 12 million people and threatening potential reversion to virulence make such vaccines unsuitable for 350 million more. Drug treatment requires long-term medication, human vaccination. Indeed, the persistence of asymptomatic in- which is expensive and highly toxic. A vaccine for leishmaniasis fection especially in immunocompromised individuals raises the has been a goal for a century, but there are still no effective vac- risk of reversion to clinical disease. Moreover, attenuations due to cines (1). The knowledge that a cured disease either due to a nat- undefined genome alterations can reduce effective protective im- ural infection or cutaneous leishmanization (1) protects the indi- munity, either they fail to persist long enough to elicit an immune vidual from reinfection and the persistence of a few parasites in the response or lack critical epitopes to evoke the protective response body can impart life-long protection against leishmaniasis, has en- (3). Alternatively, attenuation obtained through targeted genetic couraged researchers to develop live attenuated Leishmania vac- disruptions of essential growth regulating or virulence genes by cine candidates. homologous recombination is nonrevertible, hence can be safe (1). Moreover, sustained exposure of parasite Ag to the host eliminates the need of any adjuvants that are required in other nonliving vac- *Division of Emerging and Transfusion Transmitted Diseases, Center for Biologics cines (e.g., subunit vaccines). Evaluation and Research, Food and Drug Administration, Bethesda, Maryland, There are several examples of targeted gene deletions that 20892; and †Laboratory of Parasitic Diseases, National Institute of Allergy and In- fectious Diseases, National Institutes of Health, Bethesda, MD 20892 have been conducted for developing Leishmania-attenuated vaccine strains. Among the vaccine candidates studied for CL, Received for publication January 27, 2009. Accepted for publication June 2, 2009. L. major dihydrofolate reductase thymidylate synthase (dhfr- The costs of publication of this article were defrayed in part by the payment of page Ϫ charges. This article must therefore be hereby marked advertisement in accordance ts ) knockout parasites protect mice (4) but not Rhesus mon- with 18 U.S.C. Section 1734 solely to indicate this fact. keys (5). L. major deficient in surface and secreted phospho- 1 Address correspondence and reprint requests to Dr. Hira Nakhasi, Division of glycans (lpg2Ϫ), although unable to survive in sand flies and Emerging and Transfusion Transmitted Diseases, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892. E-mail address: macrophages, retained the ability to persist indefinitely in mice [email protected] and conferred protection against virulent challenge, even in the 2 Abbreviations used in this paper: CL, cutaneous leishmaniasis; MCL, mucocutane- absence of a strong Th1 response (6, 7). Over time, however, 2 Ϫ ous leishmaniasis; VL, visceral leishmaniasis; Wt, wild type; FTAg, freeze-thaw Ag; lpg (a complete gene knockout) parasites unexpectedly re- WI, week immunized; WPC, week post challenge. gained virulence (8). L. major deleted for phosphomanno- Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 mutase-protected mice, despite no increase in either effector or www.jimmunol.org/cgi/doi/10.4049/jimmunol.0900276 1814 REPLICATION DEFICIENT Leishmania INDUCES PROTECTIVE IMMUNITY memory response (9). In contrast, L. mexicana that also causes an additional confirmation of the presence of parasites in tissues, total DNA CL, deficient in cysteine proteinase genes (⌬cpa and ⌬cpb), samples obtained from infected mouse and hamster spleens were used as conferred protection in mice and hamsters against homologous templates in a Taqman-based real-time PCR. The amplification target was on the kinetoplast minicircle DNA of the parasite. The primers and meth- challenge (10, 11). Among the vaccination studies in VL, mice ods were as previously described (22), with the addition of a fluorescent immunized with a L. donovani strain deleted for biopterin trans- probe for detection. The probe had the sequence 5Ј-RAAARKKVRTRCA porter (BT1) were similarly protected (12). Recent attempts us- GAAAYCCCGT-3Ј. A Black Hole Quencher moiety is coupled to the 3Ј ing partial knockout parasites A2-A2rel gene cluster in L. do- end and Calfluor Red is coupled to a C6 linker at the 5Ј end. The degenerate letter code is according to the Nomenclature Committee of the Interna- novani (13) and SIR2 gene in L. infantum (14) as immunogens tional Union of Biochemistry (http://www.chem.qmul.ac.uk/iubmb/misc/ induced protection against virulent challenge in BALB/c mice. naseq.html). The degenerate probe allows detection of sequence variants of However, such mutants cannot be used as vaccine candidates, the minicircle found in Leishmania and is added to the reaction mixture at because they still carry wild-type (Wt) allele/s and could cause a final concentration of 1.5 pmols/␮l. To evaluate the number of Leish- disease. mania cells that were represented by a given Ct value, a standard curve was constructed by infecting macrophages ex vivo (macrophage CSF cytokine Despite limitations with most of the mutant strains, these ex- differentiated primary human monocytes) with stationary phase Leishma- periments clearly demonstrate the potential as well as the pitfalls nia. After 24 h, macrophages were trypsinized, scraped into PBS, the mac- of generating live attenuated Leishmania vaccines by targeted gene rophage cells/␮l were determined by hemocytometer, and the Leishmania deletions. Hence it is critical to develop attenuated lines through amastigotes per macrophage were determined microscopically from a smear of Diff-Quick (Baxter Healthcare Corporation) stained cells. A cal- complete gene knockouts that generate avirulent organisms that culated volume of the cell suspension was added to DirectPCR Lysis Re- persist for a brief duration but eventually are completely elimi- agent (Viagen Biotech) to produce lysates of 10 parasites/␮l, 1 parasite/␮l, nated, thereby inducing effective immunity without clinical disease and 0.1 parasite/␮l. One microliter of each concentration was used as tem- Downloaded from or the risk of reactivation. Recently, we have developed a L. do- plate in real-time minicircle PCR with four replicates to determine the novani strain completely deleted for the centrin1 gene (15). Cen- mean Ct value. trin1 is a calcium-binding basal body-associated protein involved Intracellular staining and flow cytometry in cell division in protozoan parasites like Leishmania, Trypano- Splenocytes were plated in 24-well plates and stimulated with freeze-thaw soma, and Plasmodium (16–18) and centrosome associated in Ag (FTAg) (23) or no Ag (control) in complete RPMI 1640 medium. After higher eukaryotes (19). We demonstrated that deletion of LdCen1 36 h at 37°C brefeldin A was added to the wells. After7hat37°C, cells http://www.jimmunol.org/ (LdCen1Ϫ/Ϫ) did not affect the growth of the promastigote in vitro, were blocked with anti-CD 16/32 (5 ␮g/ml) for 20 min (4°C), surface however, the growth of the mammalian-infecting, amastigote form stained with PerCP anti-CD3, allophycocyanin-Cy7 anti-CD4, and PE-Cy7 anti CD8 Abs for 30 min (each with 1/300 dilution; 4°C), fixed with Cyto- of the parasite, was blocked. These amastigotes showed failure of fix/Cytoperm kit for 20 min (room temperature), intracellular staining with basal body duplication and cytokinesis, resulting in large multinu- Alex Flour 700 anti-IFN-␥, FITC anti-TNF, Pacific blue anti-IL-2, and cleated cells in culture and ex vivo in human macrophages (15). APC anti-IL-10 or isotype-specific control for each fluorescence tagged Ab We tested this unique parasite line for its safety and protective was for 30 min (each with 1/300 dilution; 4°C). Isotype controls for each Ab used under similar conditions indicated specific binding of the test Ab. efficacy against homologous and heterologous virulent Leishmania All Abs and reagents were purchased from BD Biosciences except Pacific challenge and the immune response correlating with protection in blue anti-IL-2, which was from eBioscience. Electronic compensation was rodent animal models. performed with single-stained cells with individual mAbs used in the test by guest on September 26, 2021 samples. Cells were acquired on LSRII with DIVA software (BD Bio- Materials and Methods sciences) and analyzed by FlowJo software (Tree Star). Animals and parasites Ab responses Five- to six-wk-old female BALB/c mice from National Cancer Institute, Specific Ab responses were measured by conventional ELISA. In brief, SCID mice (BALB/c background) from The Jackson Laboratory, and ELISA plates were coated overnight at room temperature with FTAg. A 40–50 gm male Syrian golden hamsters from Charles River Laboratories serial dilution of the sera was conducted to determine the titer, which is were used in the experiments. Procedures used were reviewed and ap- defined as the inverse of the highest serum dilution factor giving an ab- proved by the Animal Care and Use Committee, Center for Biologics Eval- sorbance of Ͼ0.2. The titers for the Abs were determined using the fol- uation and Research, Food and Drug Administration. Among parasites, the lowing HRP-conjugated secondary Abs: Rabbit anti-mouse IgG (HϩL)- Ϫ Ϫ Wt, centrin1-deleted (LdCen1 / ) and centrin1 deleted but episomally HRP; Rabbit anti-mouse IgG1-HRP, Human Adsorbed, Rabbit anti-mouse Ϫ Ϫ centrin1 protein expressing line (LdCen1 / AB)ofL. donovani (15) were IgG2a-HRP; Human Adsorbed (Southern Biotechnology Associates; all used. The parasite culture procedure and the routine molecular biology with 1/1000 dilutions). SureBlue (KPL) was used as a peroxidase substrate. practices were as previously described (16). After 15 min, the reaction was stopped by the addition of 100 ␮lof1M H SO , and the absorbance was read at 450 nm. Vaccinations and challenge studies 2 4 NO quantifications In independent experiments, the mice were inoculated/vaccinated via tail vein with 3 million metacyclic cells of either L. donovani Wt, or Ld- Splenocytes or macrophages obtained from peritoneal fluid (24) were cul- Cen1Ϫ/Ϫ parasites. Infective-stage metacyclic promastigotes of L. dono- tured in complete, endotoxin-free RPMI 1640 medium (certified LPS free vani were isolated from stationary cultures by density gradient centrifuga- by the manufacturer). NO (nitrite/nitrate) production was determined for tion as described (20). Control groups (naive) corresponded to mice that the supernatants from unstimulated cultures and cultures stimulated with received a saline solution (PBS). Mice vaccinated with LdCen1Ϫ/Ϫ para- FTAg for 48 h at 37°C by the Griess reaction kit (Sigma-Aldrich). sites after different time periods were challenged via tail vein with 3 million virulent Wt L. donovani metacyclic parasites. Age-matched naive mice as Statistical analysis control groups were also similarly challenged with 3 million virulent me- Statistical analysis of differences between means of groups was determined tacyclic L. donovani parasites. In separate experiments, mice vaccinated Ͻ Ϫ/Ϫ by two-sample t test assuming unequal variance. A p value 0.05 was (with LdCen1 ) or not (naive with saline) were also challenged by in- considered as highly significant. jecting i.m. on left hind footpad with 3 million metacyclic L. braziliensis parasites. Hamsters were inoculated intracardially with 10 million meta- Ϫ/Ϫ Results cyclics of Wt or LdCen1 parasites. The hamsters vaccinated with Ld- Ϫ/Ϫ Cen1Ϫ/Ϫ or not (naive with saline) were challenged after 5 wk intracardi- Growth attenuation and limited persistence of LdCen1 ally with 10 million metacyclic virulent L. donovani parasites. After post parasites in mice challenge periods, parasite load was recorded from spleens and livers from the L. donovani-challenged mice and hamsters and footpads and lymph Centrin1-deleted L. donovani metacyclics when used to infect hu- nodes from the L. braziliensis-challenged mice by culturing the separated man macrophages in vitro or injected in susceptible BALB/c mice, host cell preparations by limiting dilutions as previously described (21). As displayed an attenuated growth (i.e., reduction in the number of The Journal of Immunology 1815

DNA from spleens of mice infected either with Wt or LdCen1Ϫ/Ϫ parasites (supplementary Table 1). Detection of the parasite mi- nicircle DNA target indicated the presence of a substantial number of parasites in both Wt-infected (6 wk) and LdCen1Ϫ/Ϫ-infected (5 wk) mice. However, the mice infected with LdCen1Ϫ/Ϫ for 12 wk showed a level equivalent to naive mice, which can be considered as a background, signifying either no LdCen1Ϫ/Ϫ parasites or sig- nificantly reduced parasite burden in mice at this time point. Hence, it appears that the parasites are present for at least 5 wk in the viscera as opposed to disappearing within just a few days. Similarly, we did not observe any LdCen1Ϫ/Ϫ parasites at 12 wk postinfection in SCID mice, indicating that their clearance is not dependent on functional T and B cells (Fig. 1b). Protective efficacy of LdCen1Ϫ/Ϫ parasites against virulent infection The ability of LdCen1Ϫ/Ϫ parasite to protect against Leishmania infection was determined in 5-wk immunized mice (WI) followed

by challenge with virulent Wt L. donovani. Spleen and liver were Downloaded from analyzed for parasite burden 4, 8, and 10 wk post challenge (WPC), measured by limiting dilution. The results showed that the immunized mice had ϳ2-log-fold reduced ( p Ͻ 0.01) parasite burden in spleen and undetectable parasite in liver compared with naive-challenged mice at 10 WPC (Fig. 1c). However, there was

no significant change in the weight of liver or spleen of immunized http://www.jimmunol.org/ or unimmunized mice. To evaluate the ability of LdCen1Ϫ/Ϫ im- munization to confer lasting protection, mice were challenged with virulent Wt L. donovani 12 or 16 wk following LdCen1Ϫ/Ϫ im- munization, and evaluated after 10 wk. The parasite burden in the spleen and liver indicated that the level of protection in both im- FIGURE 1. Avirulence and immunoprotective properties of centrin munized groups (12 WI and 16 WI) was similar to the level ob- (LdCen1) knockout L. donovani (LdCen1Ϫ/Ϫ) parasites in mice. a, Sur- served for the 5 WI group, indicating a sustained protective re- Ϫ Ϫ Ϫ Ϫ vival of parasites of LdCen1 / , LdCen1 / episomally expressing sponse after immunization (Fig. 1c). A similar result was observed Ϫ/Ϫ LdCen1 protein (add back; LdCen1 AB), and Wt in BALB/c mice. in a separate experiment where mice were challenged after 24 wk by guest on September 26, 2021 Parasite burdens from the organs of mice infected with metacyclic parasites of immunization (data not shown). No protection was observed 8 at 5 wk post infection are shown. b, Centrin knockout L. donovani does not Ϫ Ϫ WPC in mice immunized with heat killed 3 million LdCen1 / survive for long in BALB/c and SCID (BALB/c) mice. Parasite load in organs of mice infected with metacyclics of either Wt or LdCen1Ϫ/Ϫ par- metacyclics challenged 5 wk after immunization (supplementary asites were measured at 5 or 12 wk after infection. c, Protection of Ld- Fig. 1b), suggesting that live attenuated parasites were required to Cen1Ϫ/Ϫ vaccinated BALB/c mice against virulent challenge. Mice vac- induce protective immunity. Overall, the results suggest that mice Ϫ Ϫ cinated with LdCen1Ϫ/Ϫ parasites were challenged after different time immunized with LdCen1 / elicit a strong and sustained protec- periods with virulent Wt L. donovani and the challenge parasite burdens tion against the virulent challenge. from the organs were measured after different time points post challenge. The data presented are representative of two or more experiments with Induction of multifunctional Th1 effector cells correlates with Ϫ Ϫ similar results. Mean and SEM of four or more mice in each group are LdCen1 / -induced protection shown. Cha, challenged; Imm, immunized; ND, not detected; WI, wk after The involvement of IFN-␥-producing Th1 cells in immunity .p Ͻ 0.01 ,ء ;infection; WC, wk after challenge against Leishmania is described (25). We also observed an ab- solute requirement for IFN-␥ in LdCen1Ϫ/Ϫ-induced immunity. IFN-␥ knockout mice immunized with LdCen1Ϫ/Ϫ for5wk 3 infected macrophages over time; supplementary Fig. 1a), and the followed by challenge were not protected. Parasite burden from number of parasites per spleen or liver 5 wk postinfection com- these mice was similar to naive-challenged control observed at pared with control (Fig. 1a). This is the direct consequence of 10 wk post challenge (Fig. 2). To further investigate the cell- Ϫ/Ϫ centrin1 deficiency, because LdCen1 cells expressing centrin1 mediated responses induced by LdCen1Ϫ/Ϫ parasites and find a protein from a transfected plasmid were rescued for growth both in correlate of protective immunity, we measured the Th1 cyto- macrophages and in mice (supplementary Fig. 1a and Fig. 1a). To kines IFN-␥, TNF, and IL-2 produced by splenic CD4ϩ and Ϫ/Ϫ analyze the persistence of LdCen1 in mice, BALB/c mice were CD8ϩ T cells from the BALB/c mice using multiparameter flow Ϫ/Ϫ injected with LdCen1 or Wt parasites and were monitored up to cytometry. Spleen cells grown in vitro with or without freeze Ϫ/Ϫ 12 wk post infection. Few LdCen1 parasites were seen at 5 wk thaw Ags followed by multicolor staining were gated based on post infection in spleen and liver, measured by limiting dilution, forward and side scatter in FlowJo to select only the lympho- and by 12 wk, the parasites were completely cleared from both cyte population devoid of dead cells and other larger leukocytes organs (Fig. 1b). The Wt-infected mice had significant parasite (Fig. 3a, left) and further gating of the lymphocytes for the burden in these organs at both the time points (Fig. 1b). Similar CD3ϩ (Fig. 3a, middle) followed by gating of CD3ϩ in to results were obtained in a confirmatory real-time PCR study using CD4ϩ and CD8ϩ T cells (Fig. 3a, right). Seven distinct popu- lations of cytokine-producing cells were defined from the CD4ϩ ϩ 3 The online version of this article contains supplemental material. and CD8 T cells based on different combinations of IFN- ␥, 1816 REPLICATION DEFICIENT Leishmania INDUCES PROTECTIVE IMMUNITY

Induction of humoral response in the immunized mice We evaluated the humoral response in the immune-challenged mice. Sera from BALB/c mice taken 10 wk post challenge after 5, 12, or 16 immunization weeks were measured for Leishma- nia-specific IgG, IgG1, and IgG2a responses. Results indicated a higher level of all three Ab populations in the immune-chal- lenged groups compared with the naive challenged groups (Fig. 4a). Importantly, the immunization led to a selective increase in Th1-driven IgG2a Ab levels during infection ( p Ͻ 0.01). The results indicate a higher Leishmania specific humoral immune response generated by immunization with LdCen1Ϫ/Ϫ that cor- relates with the increase in the Th1 response. FIGURE 2. Lack of protection of LdCen1Ϫ/Ϫ vaccinated IFN-␥ knock- out mice against virulent challenge. IFN-␥ knockout mice vaccinated with Increased NO production in spleen cells in the LdCen1Ϫ/Ϫ parasites were challenged after 5 wk with virulent Wt L. do- immune-challenged mice novani and the challenge parasite burdens from organs were measured after Because the production of NO by macrophages is a key factor in 10 wk post challenge. Mean and SEM of four mice in each group are killing Leishmania, we determined the level of NO produced by shown. in vitro Ag restimulated splenocytes that included macrophages derived from the immune-challenged mice by ELISA. Interest- Downloaded from ingly, significantly higher ( p Ͻ 0.01) levels of the reactive NO radical (nitrite) were demonstrated in the supernatants of TNF, and IL-2 (a representative analysis is shown in Fig. 3b). splenocytes of the three immune-challenged groups (Fig. 4b). The quality of the Th1 response is based on the relative fre- We also analyzed Ag-specific release of nitrite from the cul- quency of these distinct populations (26). Five weeks after im- tured macrophages derived from the peritoneal fluids of mice. Ϫ/Ϫ ϩ munization with LdCen1 , single cytokine-producing CD4 There is a small increase in NO production in macrophages of http://www.jimmunol.org/ ϩ ␥ and CD8 T cells, making either IFN- or IL-2, were more the naive mice, suggesting a background level in this assay, as predominant than multicytokine-producing subpopulations post well as in the naive challenged mice with the addition of FTAg. immunization (Fig. 3, c and d). Similar analysis conducted with However, in the immunized, challenged mice the production of spleens of immunized mice analyzed 10 WPC showed a signif- NO in response to FTAg is significantly higher ( p Ͻ 0.01) than Ͻ icantly higher percent (2- to 10-fold; p 0.01) of the cytokine- the naive challenged mice, which clearly correlates with pro- producing subpopulations than in the naive challenged mice tection (supplemental Fig. 2). (Fig. 3, e and f). Importantly, the expansion of multifunctional Ϫ/Ϫ cells was clearly evident in the immunized mice 10 WPC. In- Safety and efficacy of LdCen1 in the hamster model

ϩ ϩ by guest on September 26, 2021 terestingly, the percent of both CD4 and CD8 T cells that Because golden Syrian hamsters are considered a more appro- make TNF alone increased significantly (up to 2-fold; p Ͻ 0.01) priate model for VL (29), we evaluated the safety and protec- Ϫ Ϫ after challenge in LdCen1 / immunized mice. The percentage tion of LdCen1Ϫ/Ϫ against L. donovani challenge in this spe- of T cells that produce Th1 cytokines increased after immuni- cies. The LdCen1Ϫ/Ϫ parasite burden in spleens, measured by zation and the increased frequency of multifunctional cells after limiting dilution, was 3-log-fold less than Wt L. donovani 5wk challenge strongly correlated with protection. after inoculation in hamsters and no LdCen1Ϫ/Ϫ parasites were We also quantitated T cells that produce IL-10, a cytokine in- found in the liver, whereas at 10 wk post immunization, no volved in the pathogenesis of VL (27). The ratio of IFN-␥ to IL-10 LdCen1Ϫ/Ϫ parasites were observed in any of the organs in serves as an additional correlate of immune protection (14). In the contrast to hamsters inoculated with virulent parasites, which ϩ restimulated CD4 T cells from the spleen, the IFN-␥/IL-10 ratio had significantly higher number of parasites in both spleen and was significantly higher in the immunized mice both at the time of liver (Fig. 5a). Further persistence of the parasites in the ham- challenge (5 WI) and after challenge (5 WI plus 10 WC) compared ster model was evaluated by performing real-time minicircle with either naive or naive-challenged controls (Fig. 3g). The re- PCR on spleen DNA samples collected more than 3 mo postin- sults thus indicate an increased IFN-␥ secretion coinciding with fection. Samples from Wt-infected hamsters gave a Ct value reduced IL-10 production among the immunized mice, indicating indicating a substantial number of parasites, while at the same a strong Th1 response that could be accountable for protective time point, LdCen1Ϫ/Ϫ-infected samples gave a Ct value equiv- immunity. alent to the uninfected hamsters. After 5 wk of immunization Recent studies in CL infection in mice indicate a role for with LdCen1Ϫ/Ϫ and challenge with virulent L. donovani, the nonregulatory T cells, which simultaneously produce IFN-␥ and immunized hamsters had significantly lower parasite burden in IL-10, for immune suppression during pathogenesis of a non- both the organs, measured by limiting dilution, compared with ϩ healing lesion (28). Hence, we also looked at IFN-␥ T cells the naive-challenged animals, with a reduction of 99.9% in that coproduce IL-10 cytokine after immunization with Ld- spleen and 99.7% in liver ( p Ͻ 0.01) (Fig. 5b). Cen1Ϫ/Ϫ. The results indicated a significantly higher percent- age of CD4ϩ T cells positive for both IFN-␥ and IL-10 in the Centrin1 KO L. donovani cross protects mice against challenge 5-wk-immunized mice than in controls (Fig. 3h). No increase of with L. braziliensis such cells in the CD4ϩ population was observed either 12 or 16 We wanted to determine whether immunization with Ld- wk after immunization (data not shown). The presence of IFN-␥ Cen1Ϫ/Ϫ could provide heterologous protection against infec- and IL-10 coproducing CD4ϩ cells early after immunization tion involving other Leishmania species. For this purpose, 5 wk (5W, a time point when LdCen1Ϫ/Ϫ parasites are still detect- LdCen1Ϫ/Ϫ immunized as well as naive BALB/c mice were able), might reflect immune modulation to reduce inflamma- challenged by s.c. inoculation in the footpad with 2 million tion-mediated host damage. metacyclics purified from L. braziliensis, the causative agent of The Journal of Immunology 1817 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 3. Multiparameter flow cytometry based analysis for distinct Th1/Th2 cytokine populations. A seven color cytometry panel was used to measure the four cytokines, IFN-␥, TNF, IL2, and IL10, with the help of three T cell membrane markers CD3, CD4, and CD8. a, The common gating steps shown in this study through FlowJo analysis are as explained in the Results section. b, Representative fluorescence intensity distributions of single, double, or triple positive cytokines (IFN-␥, TNF, and IL-2) expressing T cells in spleens of vaccinated (5 wk post vaccination) and naive mice. CD3ϩCD4ϩ or CD3ϩCD8ϩ lymphocytes from spleens showing either IFN-␥ϩ or IFN-␥Ϫ are each further separated into cells that also make either TNF, IL-2, both TNF and IL-2, or none. c–f, Flow cytometric analysis of Leishmania Ag-specific multifunctional T cells. Leishmania Ag-specific multifunctional effector cells that were measured from splenocytes of LdCen1Ϫ/Ϫ vaccinated mice at the time of challenge (5WI, 5 wk post immunization) (c and d) and after challenge (5WI plus 10WPC: 10 wk post challenge) (e and f). The percentages of CD4ϩ (c and e) and CD8ϩ (d and f) T cells producing the cytokines IFN-␥, TNF, and IL-2 and combinations of these. The analysis was conducted using multiparameter flow cytometry (as in Methods). g, Ratio of IFN-␥ to IL-10 producing CD4ϩ T cells from spleens at the time of challenge (naive and immunized (5W)) and after challenge (naive-challenged and immune-challenged (5WI plus 10WPC)). h, CD4ϩ T cells from spleens that simultaneously produce IFN-␥ and IL-10 at 5W post immunization with LdCen1Ϫ/Ϫ. The data presented are representative of two or more experiments with similar results. Mean and SEM of four or more mice in each group with error bar indicating SEM are shown. Cha, challenged; Imm, immunized; W, wk infected; .p Ͻ 0.01 ,ء ;WI, wk after immunization; WPC, wk after challenge

MCL. The naive, challenged mice developed progressive le- lenged mice showed significantly lower parasite burdens both in sions during the 7 wk of observation, whereas the immunized the footpads and lymph nodes compared with the controls (Fig. mice developed significantly smaller lesions (Ͻ3-fold; p Ͻ 5d), with a 99.9% reduction in both the tissues ( p Ͻ 0.02; p Ͻ 0.01) throughout the 7 wk (Fig. 5c). The immune-chal- 0.01). A similar type of experiment conducted to examine cross 1818 REPLICATION DEFICIENT Leishmania INDUCES PROTECTIVE IMMUNITY

FIGURE 5. Avirulence and immuno-protective properties of LdCen1Ϫ/Ϫ parasites in hamsters. a, Parasite load in organs of hamsters infected with either Wt or LdCen1Ϫ/Ϫ metacyclic parasites was measured at 5 and 10 wk after infection. b, Protection of the LdCen1Ϫ/Ϫ vaccinated Downloaded from hamsters against virulent challenge. Hamsters vaccinated with LdCen1Ϫ/Ϫ parasites were challenged after 5 wk of immunization with virulent Wt L. donovani and the challenge parasite burdens from the organs were mea- sured after 4 wk. c, Cross-protection of BALB/c mice vaccinated with LdCen1Ϫ/Ϫ against heterologous challenge with L. braziliensis. Five wk post vaccinated mice were infected with metacyclics of L. braziliensis in- jecting i.m. in the left-hind footpad of each mouse. Graph shows footpad http://www.jimmunol.org/ swelling due to infection in the injected footpad calculated by measuring the difference in the footpad size between the two hind footpads. d, Parasite numbers per organ of infected footpad or draining lymph nodes from 7 wk postchallenge with L. braziliensis are shown. The data presented are rep- resentative of two experiments with similar results. Mean and SEM of four animals in each group are shown. Cha, challenged; Imm, immunized; ND, .p Ͻ 0.01 ,ءء p Ͻ 0.02 and ,ء ;not detected; W, wk after infection FIGURE 4. a, Humoral response in the virulent challenged mice vac- Ϫ Ϫ cinated with LdCen1 / parasites. ELISA measurement of IgG, IgG1, and by guest on September 26, 2021 Ϫ/Ϫ IgG2a Abs in sera from the mice immunized with LdCen1 parasites for To evaluate it as a vaccine candidate, the LdCen1Ϫ/Ϫ parasite 5, 12, or 16 wk or naive before challenge with virulent L. donovani. Sera was tested both in susceptible BALB/c mice and Syrian hamsters. were collected 10 wk post challenge. b, Leishmania Ag specific stimulation Ϫ Ϫ LdCen1 / parasites showed persistence of low numbers of par- of NO synthase (NOS2) in the splenocytes of naive, naive challenged and Ϫ/Ϫ asites both in mice and hamsters for at least 5 wk. Ten (both in LdCen1 immunized and Wt challenged mice. The activity of NOS2, indicated by the amount of released nitrite (NO) in the splenocyte super- mice and hamsters) or 16 wk postinfection and in some cases even Ϫ/Ϫ natants was measured by the Griess reaction. The data presented are rep- longer periods (24 wk; data not shown), LdCen1 parasites were resentative of two experiments with similar results. Mean and SEM of three not detected in liver or spleen of mice, suggesting the complete or more mice in each group are shown. Cha, challenged; Imm, immunized; clearance demanded of a safe vaccine candidate. Similar observa- FTAg, Freeze-thaw Ag; WI, wk after immunization; WPC, wk after chal- tions in SCID mice lacking both T and B cells reinforce its safety .p Ͻ 0.01. even in the immune compromised host ,ء ;lenge While searching for correlates of protection against homologous challenge, we found that LdCen1Ϫ/Ϫ immunized mice developed an increased percent of Leishmania specific CD4ϩ and/or CD8ϩ T Ϫ Ϫ protection of LdCen1 / against L. major showed a moderate cells expressing Th1 cytokines (IFN-␥, TNF, and IL-2) either sin- but delayed protection (data not shown). gly or in multiple combinations. Multifunctional effector cells as- sociated with protection have been described by others in mice Discussion vaccinated with Leish-111f recombinant polyproteins plus adju- In pursuit of a candidate live attenuated vaccine, we generated a L. vant (30) and with Leishmania MML protein expressed in a rep- donovani parasite that had a complete deletion for a cell division lication-defective adenovirus (26). We found an especially strong gene, centrin1 (LdCen1Ϫ/Ϫ) (15). We have demonstrated that increase of TNF-producing T cells, reinforcing prior data suggest- LdCen1Ϫ/Ϫ attenuation is due to the failure of cytokinesis, result- ing that TNF along with IFN-␥ secretion are important cytokines ing in multiplication of cellular organelles, cell enlargement, and for protection in vivo (26). We also observed an increased IFN- eventual programmed cell death. We think that the genetically tar- ␥/IL-10 ratio among the CD4ϩ T cells in the LdCen1Ϫ/Ϫ vacci- geted and defined attenuation reduces the risk of reversal to viru- nated mice both at the time of and after challenge, revealing an- lence, a concern generally raised for attenuated organisms that are other correlate of protection. A similar polarization to an increased created by random genomic mutations. LdCen1 deletion specifi- IFN-␥ to IL-10 ratio in the splenocyte supernatant measured by cally attenuated the amastigote stage of the parasite that replicates ELISA after L. infantum challenge in mice immunized with inside macrophages, and has no effect on the growth of the pro- SIR2ϩ/Ϫ L. infantum was observed (14). We also observed an mastigote form. This could be advantageous to grow large quan- increase of CD4ϩ T cells that coproduce IFN-␥ and IL10 during tities of parasites for vaccine trials. the initial period (5W) of infection with LdCen1Ϫ/Ϫ, suggesting The Journal of Immunology 1819 possible immune modulation at this time as also observed previ- cies of Leishmania has been documented in animal model studies ously, during L. major infection in mice (28). In the L. major case, (37–39). In mice, experimental vaccination using dp72 protein iso- the nonregulatory T cells (CD4ϩCD25ϪFoxp3Ϫ) that were the lated from L. donovani cross-protected against L. major infection source of IL-10 (most of them also produced IFN-␥) were immu- (40) and immunization with exogenous Ags (LmSEAgs) of L. ma- nosuppressive during protozoan infection for the reduction of in- jor cross-protected against L. donovani (41). Our results show that fection mediated damage to host cells. Hence, the LdCen1Ϫ/Ϫ im- LdCen1Ϫ/Ϫ-immunized mice were cross-protected to a high de- munization may be facilitating a desirable balance in the immune gree against a heterologous challenge with L. braziliensis, that response allowing brief parasite persistence that facilitates protec- causes MCL, and to some degree against L. major that causes CL. tion without causing host damage. Therefore, our results confirm that the live attenuated parasites also In the present study, we observed a robust Th1-specific serum confer resistance to infection with other species. Ab (IgG2a) response in the immune-challenged mice, further sup- In the literature, several routes of administration of Leishmania porting the observation that LdCen1Ϫ/Ϫ induces a generalized Th1 vaccine candidates have been studied with varied degree of pro- type response. In addition, the increased release of NO observed in tection (reviewed in Ref. 42). In the present study, we have used the splenocyte culture derived from the immune-challenged mice the i.v. route in mice and intracardial route in hamster, however we coincided with the increase of Leishmania specific T cells produc- recognize that in general a recommended route of administration ing IFN-␥, TNF, and IL-2 cytokines. NO production induced by of a vaccine in humans is either intradermal or i.m. Future studies Th1 cytokines is a main leishmanicidal mechanism of murine mac- in our laboratory will focus on defining the ideal route of admin- rophages (31, 32). Thus, in the LdCen1Ϫ/Ϫ-immunized mice, NO istration for optimal protection for LdCen1Ϫ/Ϫ parasites. production by macrophages that are stimulated by Th1 cytokine In summary, this study demonstrates that in mice and hamsters, Downloaded from producing T cells correlates with the control of infection. the live attenuated LdCen1Ϫ/Ϫ is highly immunogenic and confers Importantly, immunization with LdCen1Ϫ/Ϫ protected both a significant degree of protection against L. donovani and also is mice and hamsters against virulent homologous challenge, as in- capable of inducing cross protection against L. braziliensis. The dicated by significantly reduced parasite burdens in the organs of vaccine-elicited parasite Ag-specific Th1 responses coinciding the immune-challenged mice and hamsters compared with naive with a robust Ab response and NO production, all strongly corre-

controls. The protective response was sustained for up to 24 wk lated to a sustained protection. This report thus presents a well http://www.jimmunol.org/ after immunization. Complete elimination of wild-type parasites characterized amastigote specific, live attenuated L. donovani can- from the livers of LdCen1Ϫ/Ϫ immunized mice after virulent chal- didate vaccine that has been evaluated for its in vivo persistence, lenge, is superior to the protection induced by L. donovani immunological response, and protective (against homologous and BT1Ϫ/Ϫ, the only other complete gene knockout strain reported to heterologous challenges) efficacy. target VL, which reduced parasite level in liver by 75% of the level in naive challenged mice (12). The dhfr-tsϪ auxotropic L. major Acknowledgments line was safe in both mice and rhesus monkeys but protected only We acknowledge Kimberly Beacht for technical assistance and Sanjai Ku- Ϫ the mice (4, 5). Because the immune responses due to dhfr-ts mar and Alain Debrabant for valuable suggestions. were not studied in those animals, it would be difficult to know if by guest on September 26, 2021 there were weakness in the mouse immune response that could Disclosures have predicted failure in the monkey. However, the quality of The authors have no financial conflict of interest. LdCen1Ϫ/Ϫ-induced immune response in mice and protection in mice and hamsters encourage us to proceed with immune studies References in higher animal models. The level of protection achieved by 1. Selvapandiyan, A., R. Duncan, A. Debrabant, N. Lee, G. Sreenivas, P. Salotra, LdCen1Ϫ/Ϫ was similar to the protection due to L. infantum and H. L. Nakhasi. 2006. Genetically modified live attenuated parasites as vac- ϩ/Ϫ L. infantum ϩ/Ϫ cines for leishmaniasis. Indian J. Med. Res. 123: 455–466. SIR2 in mice (14). Because SIR2 is a partial 2. Silvestre, R., A. Cordeiro-da-Silva, and A. Ouaissi. 2008. 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Sci. USA 92: 10267–10271. There are several possible explanations for the protection by 5. Amaral, V. F., A. Teva, M. P. Oliveira-Neto, A. J. Silva, M. S. Pereira, Ϫ/Ϫ E. Cupolillo, R. Porrozzi, S. G. Coutinho, C. Pirmez, S. M. Beverley, and LdCen1 parasite vaccination. First, there could be residual par- G. Grimaldi, Jr. 2002. Study of the safety, immunogenicity and efficacy of at- asite burden (beyond the detection sensitivity of our methods, tenuated and killed Leishmania (Leishmania) major vaccines in a rhesus monkey (Macaca mulatta) model of the human disease. Mem. Inst. Oswaldo Cruz 97: which would allow continued presence of Leishmania-specific ef- 1041–1048. fector cells and maintain anti-Leishmania immunity (21, 33). Sec- 6. Spath, G. F., L. F. Lye, H. Segawa, D. L. Sacks, S. J. Turco, and S. M. Beverley. ond, vaccination with LdCen1Ϫ/Ϫ parasites could lead to genera- 2003. Persistence without pathology in phosphoglycan-deficient Leishmania ma- jor. Science 301: 1241–1243. tion of a central memory T cell response after the KO parasites are 7. Uzonna, J. E., G. F. Spath, S. M. Beverley, and P. Scott. 2004. Vaccination with cleared that could develop into an effector memory T cell response phosphoglycan-deficient Leishmania major protects highly susceptible mice from upon challenge, and provide protection (34). Third, there could virulent challenge without inducing a strong Th1 response. J. Immunol. 172: 3793–3797. be Ag persistence in peripheral tissues long after the parasites are 8. Spath, G. F., L. F. Lye, H. Segawa, S. J. Turco, and S. M. Beverley. 2004. cleared. These persistent Ag “depots” may contribute to specific Identification of a compensatory mutant (lpg2-REV) of Leishmania major able to survive as amastigotes within macrophages without LPG2-dependent glycocon- memory T cells through the activation of naive T cells as has been jugates and its significance to virulence and immunization strategies. Infect. 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