6 mice against cutaneous leishmaniasis

Vaccine 21 (2003) 3534–3541

Interferon-gamma-inducing oral vaccination with Leishmania amazonensis antigens protects BALB/c and C57BL/6 mice against cutaneous leishmaniasis Eduardo Fonseca Pinto, Marcelle de Mello Cortezia, Bartira Rossi-Bergmann∗ Instituto de Biof´ısica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, 21.949-900 Rio de Janeiro, Brazil Received 15 January 2003; received in revised form 13 May 2003; accepted 20 May 2003

Abstract The induction of oral tolerance against disease-inducing antigens has emerged as a feasible strategy to prevent immunopathologies. In this study, we investigated the effect of oral immunization with whole antigens of Leishmania amazonensis promastigotes (LaAg) on murine cutaneous leishmaniasis. We found that two oral doses with 100 ␮g LaAg rendered BALB/c and C57BL/6 mice more resistant against subsequent infection with L. amazonensis. The oral vaccine also partially protected BALB/c mice against Leishmania major infection. Unlike the oral route, hepatic immunization was without effect, indicating a requirement for antigen passage through the gut mucosa. Oral LaAg significantly impaired the capacity of infected BALB/c mice to mount a disease-associated hypersensitivity response, compatible with peripheral tolerization. Both IFN-␥ and IL-10, but not IL-4 were greatly elevated in the mesenteric lymph nodes whereas only IFN-␥ was increased in the peripheral lymph nodes, compatible with a TH1 cytokine response. Gamma delta TCR+ T cells may be an important component in antigenic sensitization of the gut mucosa since their depletion during oral immunization reverted protection. These results demonstrate for the first time the feasibility of using the oral route of immunization to induce protection against cutaneous leishmaniasis using a crude parasite antigen. © 2003 Elsevier Ltd. All rights reserved. Keywords: Leishmania; Oral vaccine; Tolerance

1. Introduction Leishmania spp. are pathogenic protozoa that cause a wide spectrum of clinical manifestations in man and other mammalian hosts ranging from single cutaneous ulcers to mutilating facial lesions and fatal visceral infection. The disease is endemic in 88 countries, with an estimated 2 million new cases per year [1]. Despite the recent advances in new antileishmanial compounds, the first-line therapy to all forms of the disease still requires potentially toxic and painful multiple injections with pentavalent antimonials [2]. The problem is further aggravated by the surge of antimonial-resistance in some endemic areas [3]. The development of a vaccine against leishmaniasis is a long-term goal in both human and veterinary medicine. In the past decade, various subunit and DNA antigens have been identified as potential vaccine candidates in experimental animals [4], but none have so far been approved for ∗ Corresponding author. Tel.: +55-21-2260-6963; fax: +55-21-2280-8193. E-mail address: [email protected] (B. Rossi-Bergmann).

0264-410X/$ – see front matter © 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0264-410X(03)00427-4

human use. Vaccine formulation with killed parasites is still attractive in terms of cost. Intramuscular or intradermal injections with killed Leishmania amazonensis promastigotes were shown to convert Montenegro skin test and to induce IFN-␥ responses in a percentage of vaccinated humans [5,6], but induction of protection is conflicting and remains to be ascertained. Experiments in mice [7,8] and monkeys [9] have suggested that such formulation contains disease-inducing antigens, as i.m., i.d. or s.c. inoculation with whole L. amazonensis promastigote antigens actually resulted in the exacerbation of subsequent infections. Such harmful antigens may be important for parasite survival in the host possibly by diverting protective immune responses. Mucosal vaccination using disease promoting antigens has been considered as a novel strategy to induce protection against autoimmune and allergic pathologies [10]. The notion that immunological tolerance may provide the host with a protective mechanism against infectious diseases was demonstrated in susceptible mice transgenically expressing LACK, a TH2-inducing antigen, in the thymus. Unlike wild-type mice, which developed overwhelming lesions associated with TH2 responses, LACK-expressing mice

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produced increased IFN-␥ and were resistant to Leishmania major infection [11]. More recently, tolerization of post-thymic, mature parasite-specific T cells could also be accomplished in the periphery after nasal administration of L. major LACK antigen, but conjugation with the adjuvant cholera toxin ␤ subunit was necessary [12]. Such treatment delayed the onset of lesion development in infected mice and reduced parasite burden in the skin and draining lymph nodes of infected mice. Adjuvant requirement may be a draw back for vaccine use in human. Here, we use the oral route of immunization with a crude disease-inducing leishmanial antigen to evaluate the feasibility of inducing protection against cutaneous leishmaniasis through mucosal tolerance. 2. Materials and methods 2.1. Mice BALB/c and C57BL/6 mice were originally purchased from Jackson Laboratory (Bar Harbor, Maine). They were bred and maintained at our own facilities, using sterilized bedding, filtered water and pelleted food. Female animals were used at 4–6 weeks of age. 2.2. Parasites L. amazonensis (designation MHOM/BR/75/Josefa strain) and L. major LV 39 (designation MRHO/SU/59/P strain) promastigotes were used. Parasites were routinely isolated from mouse lesions and maintained as promastigotes in Dulbecco-modified Minimum Essential Medium (D-MEM, Sigma Chemical Co., USA) containing 10% heat inactivated fetal calf serum (HIFCS, Cultilab, Brazil), 50 U/ml penicillin and 50 ␮g/ml streptomycin (Cultilab, Brazil) at 26 ◦ C. 2.3. Antigens (LaAg) L. amazonensis promastigotes at early stationary growth phase were washed three times by centrifugation at 1300 × g for 10 min in PBS. The pellet was resuspended at 2 × 108 ml−1 PBS and submitted to three cycles of freezing and thawing. The lysate so prepared was termed LaAg. One milliliter of LaAg was shown to contain 1540 ␮g protein, as measured by the Lowry assay. All samples were kept at −20 ◦ C until required. 2.4. Immunization with LaAg For oral immunization, BALB/c or C57BL/6 mice, as appropriate, were fasted for 3 h before receiving 100 ␮g LaAg in 200 ␮l PBS by intragastric gavage on days −14 and −7 of infection. For s.c. immunization, animals were injected with 25 ␮g LaAg in 100 ␮l PBS in the base of the tail using

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the same time schedule. Alternatively, mice received a single dose of 100 ␮g LaAg in 20 ␮l PBS by the cecal vein on day −7 of infection. For this, mice were anesthetized with Nembutal and a small abdominal incision was made to expose the cecum. LaAg or control PBS was injected through the cecal vein using a 30-gauge needle. After injection, the needle was rapidly withdrawn, and hemostasis was secured by gentle pressure with gel-foam. Complications were seen in less than 10% of mice, and these were excluded from analysis because of post-injection hemorrhage. 2.5. Depletion of γδ-TCR+ T cells Where applicable, mice received 1 mg anti-mouse ␥␦TCR (clone UC7-13D5, BD Pharmingen, USA) or 1 mg control hamster IgG (clone A19-3, BD Pharmingen) by i.p. route in 100 ␮l PBS 2 days before each LaAg oral immunization as above. Percentage staining by FACS of lymphocyte-gated spleen cells from untreated or anti-␥␦-TCR-treated mice 2 days after the second antibody injection was 4.9 ± 0.1 and 0.0 ± 0.0, respectively. Animals were infected with L. amazonensis 7 days after the last dose of oral LaAg as described above. 2.6. Infection Seven days after the last immunization, animals were infected in the hind footpad with 3×106 washed promastigotes of L. amazonensis at early stationary growth phase. Lesion sizes were measured with a dial caliper (Mitutoyo, Brazil) every 4–5 days and expressed as the difference between the thickness of contralateral uninfected footpad. Alternatively, for the determination of parasite load in the lesions, animals were infected in the same way with L. amazonensis rendered fluorescent by transfection with green fluorescent protein [13]. Briefly, each infected feet were cut off and homogenized in 2 ml PBS using a tissue grinder. After removal of tissue debris by gravity sedimentation for 10 min, 200 ␮l of two-fold dilutions of the cell suspensions were transferred in triplicate to black microplates and fluorescence read in a plate-reader fluorometer (Fluoroskan, USA) at 435 ␩m excitation/538 ␩m emission. 2.7. Cytokines The mesenteric and popliteal lymph nodes were excised from 35 days infected animals and single-cell suspensions prepared in Dulbecco-modified Minimum Essential Medium (D-MEM, Sigma Chemical Co., USA) containing 10% HIFCS and antibiotics (50 U/ml penicillin + 50 ␮g/ml streptomycin). Cells were plated in triplicate in 24-well culture plates at 4 × 106 ml−1 and stimulated with 2.5 ␮g/ml Concanavalin A (Con A, Sigma Aldrich, USA) for 48 h at 37 ◦ C with 5% CO2 in the atmosphere since L. amazonensis antigens fail to activate lymphocyte responses in vitro (unpublished). The levels of IFN-␥, IL-4 and IL-10

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were measured in two-fold dilutions of the supernatants by ELISA. The levels of cytokines were determined against standard curves using recombinant murine cytokines and antibodies from R&D Systems (USA).

*

2.8. Jones–Mote hypersensitivity reaction Infected mice were injected in the contra lateral footpad with 20 ␮g LaAg in 20 ␮l PBS. Footpad swelling was measured with a dial caliper (Mitutoyo, Brazil) on the indicated times and expressed as the difference between the thickness before and after injection.

Statistical significance (P < 0.01 or P < 0.05) of differences between immunized and non-immunized groups of mice was determined by Student’s t test.

3. Results 3.1. Comparison between oral, hepatic and subcutaneous pre-immunization with LaAg on L. amazonensis infection Previous studies have demonstrated that s.c. immunization with whole irradiated L. major promastigotes promotes increased lesion growth in BALB/c mice infected with homologous parasites [8]. In this work, we show that greater predisposition to infection after s.c. immunization is also achieved using L. amazonensis whole promastigote antigens (LaAg) (Fig. 1, top). However, the opposite effect is seen when LaAg is given by the oral route. LaAg delayed the onset and reduced the size of lesions with doses as small as 100 ␮g (Fig. 1, middle). Increasing oral doses up to 1000 ␮g did not promote significant increase in protection (data not shown). To evaluate the role of gut-associated lymphoid tissue (GALT) and/or passage through the digestive system in oral protection, 100 ␮g LaAg was injected on day −7 directly into the cecal vein for primary hepatic uptake. No significant (P > 0.05) reduction in lesion sizes was observed following this process (Fig. 1, bottom), suggesting that passage through the gut is necessary for effective protection. 3.2. The effectiveness of oral vaccination with LaAg is extensive to other parasite and mouse species Mice of different genetic backgrounds display differential susceptibility to L. amazonensis [14]. To evaluate whether the oral effectiveness of LaAg is associated with the host genetic background, C57BL/6 mice were submitted to the same vaccination protocol as used for BALB/c mice. As seen in Fig. 2, both vaccinated and unvaccinated groups of C57BL/6 mice developed a milder infection in the first few weeks of infection, consistent with a relatively more resistant

Lesion size (mm)

2.9. Statistical analysis

* **

Days post infection Fig. 1. Effect of subcutaneous, oral and hepatic pre-immunization with LaAg on subsequent infection with L. amazonensis. BALB/c mice were immunized with LaAg (䊉) as follows: 25 ␮g on days −14 and −7 of infection by subcutaneous route (top), 100 ␮g on days −14 and −7 by oral route (middle); or 100 ␮g on day −7 in the liver via the cecal vein (bottom). Controls received PBS (䊊). Animals were infected on day 0 with L. amazonensis in the footpad and the lesion sizes scored on the indicated days. Results representative of three independent experiments. Mean ± S.D., n = 5, (∗) P ≤ 0.05.

phenotype of this strain as compared with BALB/c mice in Fig. 1. From day 40 of infection on, the vaccinated animals were able to significantly control lesion growth, unlike the unvaccinated group in which the lesions grew steadily. This finding indicates that the effectiveness of oral LaAg is not

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to L. amazonensis, and may induce cross-protection against other Leishmania species.

PBS LaAg Lesion size (mm)

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3.3. Pre-immunization with oral LaAg depresses the capacity of mice to mount a disease-associated Jones–Mote hypersensitivity response

* * *

Days post infection Fig. 2. Effectiveness of oral LaAg in C57BL/6 mice. C57BL/6 mice received 100 ␮g of oral LaAg (䊉) or PBS (䊊) on days −14 and −7 of infection. Animals were infected with L. amazonensis on day 0 as for Fig. 1 and the lesion sizes scored on the indicated days. Mean ± S.D., n = 5, (∗) P < 0.01.

restricted to BALB/c and may apply to mice of different genetic backgrounds. To test for cross-protection, BALB/c mice were infected with L. major following oral immunization with LaAg. It was interesting to note that the protective effect of pre-immunization with oral LaAg during infection was long-lasting, as observed by the capacity of animals to withhold lesion growth for more than 100 days of infection in the L. major model of infection (Fig. 3). Although the L. major strain used was not very virulent, this finding indicates that the efficacy of this oral vaccine is not restricted

The so-called Jones–Mote response, unlike the classical delayed-type hypersensitivity (DTH) response which peaks at 48 h, is associated with the inability of BALB/c mice to fight infection with L. major [15]. Here, we show that infection of BALB/c mice with L. amazonensis also promotes the same pattern of hypersensitivity response which peaks at 18 h upon s.c. challenge with LaAg. A significant swelling was first detected 10 h after antigen administration. This peaked at 16–19 h and was undetectable at 40 h (Fig. 4). This finding indicates that the oral LaAg vaccine systemically suppresses the capacity of immunized animals to respond with a hypersensitivity response which is associated with the susceptible phenotype in murine cutaneous leishmaniasis. 3.4. Pre-immunization with oral LaAg induces a balanced cytokine response in both peripheral and draining lymph nodes during infection To more specifically determine how the oral vaccination was affecting the immune responses during infection, the levels of the main TH1 (IFN-␥) and TH2 (IL-4 and IL-10)-type cytokines were measured in the gut mucosa- and in the lesion-draining lymph nodes 35 days after BALB/c

PBS LaAg

* *

swelling (mm x 10)

Lesion size (mm)

2

PBS 1.5

LaAg

1

0.5

* *

* *

0

hours Days post infection Fig. 3. Oral LaAg is effective against L. major infection. BALB/c mice received 100 ␮g of oral LaAg (䊉) or PBS (䊊) on days −14 and −7 of infection. Animals were infected with L. major on day 0 as for Fig. 1 and the lesion sizes scored on the indicated days. Mean ± S.D., n = 5, (∗) P < 0.01.

Fig. 4. Effect of oral LaAg on the Jones–Mote hypersensitivity response. BALB/c mice received 100 ␮g of oral LaAg (䊉) or PBS (䊊) on days −14 and −7 of infection and were infected with L. amazonensis on day 0 as for Fig. 1. On day 9 of infection animals were challenged in the contralateral footpad with LaAg and the kinetics of the swelling response scored in the following hours. Mean ± S.D., n = 4–5, (∗) P < 0.01.

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mouse infection with L. amazonensis. As shown in Fig. 5, the production of all cytokines, particularly of the TH2-type, were higher in peripheral than in mesenteric lymph nodes during infection of unvaccinated animals (white bars). Oral

ng/ml

* *

vaccination (black bars) resulted in significant increase in the production of IFN-␥ in both peripheral and mesenteric lymph nodes. As to the TH2-type cytokines, the vaccination induced a moderate decrease in the production of IL-4 by the peripheral lymph nodes, whereas the levels of IL-10 were increased in the mesenteric lymph nodes. Those findings indicate that the cells in the mesenteric lymph nodes remained sensitized even after several weeks of antigen ingestion. Moreover, they are suggestive that the reversion of cytokine pattern in the peripheral lymph nodes favoring a TH1 response, may help explain the protective systemic effect of the oral LaAg. 3.5. The protectiveness of oral LaAg is associated with γδ-TCR+ T cells

PL

ML

ng/ml

*

PL

ML

␥␦-TCR+ T cells are key cells involved in oral and hepatic tolerization with allogeneic cells, resulting in enhanced skin graft survival in mice [16]. Since we observed reduced Jones–Mote-type hypersensitivity responses and increased production of IL-10 by mesenteric lymph nodes in infected animals following oral immunization with LaAg, compatible with oral tolerance, we attempted to evaluate the role of those cells in the protective effect of oral LaAg on cutaneous leishmaniasis. As seen in Fig. 6, the lesion size of mice on the 110th day of infection was significantly enhanced when the oral immunization was preceded by depleting anti ␥␦-TCR treatment. The effect was not due to increased local inflammation as confirmed by the equivalent increase in parasite load in the infected tissue. Pilot experiments demonstrated a complete depletion of ␥␦-TCR+ T cells in the spleen following the two doses of antibody (Section 2.5), indicating that fluorescence units

mm

*

ng/ml

*

*

PL

ML

Fig. 5. Cytokine production in peripheral and mesenteric lymph nodes. BALB/c mice (five per group) were pre-immunized with oral LaAg (black bars) or received only oral PBS (white bars) before infection with L. amazonensis. On day 35 of infection, the levels of IFN-␥, IL-4 and IL-10 produced by peripheral (PL) and mesenteric (ML) lymph node cells in the culture supernatants was measured by ELISA. Mean ± S.D. of triplicate samples, (∗) P < 0.05.

Lesion size

Parasite load

Fig. 6. Effect of ␥␦-TCR+ T cell depletion on the protectiveness of oral LaAg. BALB/c mice were injected i.p. with anti-␥␦-TCR (black bars) or control antibody (white bars) 2 days before each oral dose of LaAg. Seven days after LaAg they were infected with fluorescent L. amazonensis and 110 days later the lesion size was scored in millimeters and the parasite load in the infected feet scored as fluorescence units. Mean±S.D., n = 3–4, (∗) P < 0.01.

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the cells were effectively removed prior to the oral immunization. This finding supports the notion that ␥␦-TCR+ T cells are important for the protectiveness achieved with the pre-immunization with oral LaAg against cutaneous leishmaniasis.

4. Discussion Earlier studies attempting to vaccinate BALB/c mice with crude L. major leishmanial antigen delivered by the subcutaneous, intradermal or intramuscular routes proved disappointing as such vaccines invariably exacerbated subsequent homologous infection [7,8]. By using the s.c. route, we observed here that the deleterious effect of crude L. major is extensive to L. amazonensis (Fig. 1). Our finding is also in accordance with the work by Kenney et al. who showed that in Rhesus monkeys the s.c. pre-immunization with heat-killed L. amazonensis promastigotes exacerbated the lesions produced by L. amazonensis infection [9]. In that study, protection was achieved only if the antigen was delivered with both rIL-12 and alum as adjuvants. Together, those findings indicate that killed promastigotes of L. major and L. amazonensis contain disease-inducing antigens. Considerable interest has been focused recently on the possibility of inducing oral tolerance against diseaseinducing antigens to treat immunopathologies by using the mucosal route for antigen delivery. Although clinical trials on TH1-driven diseases such as autoimmune multiple sclerosis and rheumatoid arthritis have yielded conflicting results [17], TH-2-driven allergic diseases may prove more promising [18–20]. The use of a tolerogenic approach to develop protective immunity in murine leishmaniasis has been documented by Liew and co-workers who showed the protective effect of intravenous administration of irradiated parasites before infection with L. major of susceptible or resistant mice [21,8], a protocol which reduce the frequency of Leishmania-specific T cells during infection [22]. The notion that immunological tolerance may provide the host with a protective mechanism against leishmaniasis was elegantly illustrated by studies in transgenic mice expressing a deleterious parasite antigen in the thymus. Whereas mice from a susceptible BALB/c background normally develop an early TH2-driven IL-4 response and ultimately succumb to their infection with L. major, mice rendered tolerant by transgenic expression in the thymus of LACK, a TH2-inducing parasite surface antigen, fail to produce this early response and resolve their infection [11]. Even in resistant mice, in which the effects of responsiveness to LACK are eventually overcome, artificial removal of the LACK-specific T-cell population by expression of LACK in the thymus accelerates the normal rate of cure [23]. However, those authors failed to induce protection in L. major infected BALB/c mice when using either oral or nasal administration with recombinant LACK at doses as high as 8 mg [12]. In that study, only when the recombinant protein

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was conjugated with the mucosal adjuvant cholera toxin ␤ subunit was the nasal vaccination effective in delaying the onset of infection and reducing parasite burden. In the present study, we describe for the first time that oral immunization with sole leishmanial antigens can protect mice against cutaneous leishmaniasis. It was interesting to observe that such vaccination effectively induced cross-protection against another Leishmania species and that it is not restricted by the host genetic background, suggesting that it may have a broad spectrum of application. The presence of particulate antigens in the LaAg may help explain the lack of adjuvant requirement, which is greatly advantageous, since there is as yet no mucosal adjuvant approved for human use [24]. The use of the oral route for the administration of attenuated Salmonella typhimurium expressing the leishmanial antigen gp63 to protect BALB/c mice against L. major infection was described before by Xu et al. [25]. However, in that study the role of mucosal immunity was not in question, as the antigens were actually being delivered parenterally by the invasive bacteria which remained alive for 3 weeks in the lymphoid organs. Apparently, 1–2% of orally administered proteins may enter the blood in its native form [26]. In our work, we found that if the LaAg reached the liver in its native form, the vaccination was ineffective (Fig. 1). This finding suggested that antigen processing or presentation in the gut mucosa was an important event for oral LaAg efficacy. The observation that ␥TCR+ cells depletion at the time of oral pre-immunization significantly increased the lesion size and parasite load during subsequent infection (Fig. 6) suggests that those cells may be important for oral LaAg-induced protection. Although studies using TCR ␥ deficient-C57BL/6 mice demonstrated that those cells are not important for the control of L. major infection [27], the possibility that ␥TCR+ cells depletion could affect the course of infection should be considered since bi-weekly treatment of BALB/c and CBA/J mice with anti-␥ TCR throughout the infection with L. major may eventually promote enhanced lesions [28]. Nonetheless, we observed no effect of pre-infection depletion of ␥TCR+ T cells on the course of L. amazonensis infection in non-immunized animals (data not shown). It has been long known that mucosally induced systemic tolerance depends on an intact epithelial barrier [29,30] which provides the epithelium with a central role in tolerance induction. Since murine intestinal ␥TCR+ T cells are an important component of mucosal immunity, and have been associated with mucosal tolerance [31–33], it is tempting to speculate that the protection achieved by oral LaAg is associated with those cells. Mucosal uptake of antigens may result in the development of tolerance or immunity involving the production of TGF-␤ TH1 or TH2 responses, the decision being mainly determined by the nature and physico-chemical form of the antigen [34]. The mechanisms underlying the oral protection with LaAg could not be clearly established here, but the observation that the disease-associated [15,35] Jones–Mote

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hypersensitivity reaction (Fig. 4) and IL-4 (Fig. 5) are diminished whereas IFN-␥ is increased (Fig. 5) in the peripheral lymph nodes is indicative of selective tolerization of TH2 responses favouring the expansion of a TH1-type response with protection against infection. Whether or not the TGF-␤ was modulated is not known, but the increased IL-10 in the mucosal-draining lymph nodes may indicate a role for this cytokine in peripheral tolerance induction [36]. Since IL-10 levels were not increased in the lesion-draining lymph nodes, its likely that activation of macrophages by elevated IFN-␥ prevailed in the site of infection. We found that the oral vaccine is likely to be effective only as a prophylactic measure, as the ongoing L. amazonensis infection of BALB/c mice was not controlled when the first oral dose was given 5 days after infection (data not shown). It is worth of note that the LaAg is similar in composition to a vaccine (Leishvacin® ), which has passed phases 1 and 2 clinical trials for safety and immunogenicity in human volunteers after systemic administration [37]. The work presented here points to another application of such vaccine already produced in industrial scale and most importantly, demonstrates the feasibility of vaccination against cutaneous leishmaniasis using the oral route, which is the most convenient and acceptable means of vaccine delivery.

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