ISCOMs vaccine against experimental leishmaniasis

PII: SO264-410X(97)00308-3

Vaccine, Vol. 16, No. 9/l 0. pp. 885-892, 1998 0 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0264-410X/98 $19+0.00

ELSEVIER

ISCOMs vaccine against experimental leishmaniasis Georgia

Papadopoulou*t,

Evdokia

Karagouni”

and Eleni Dotsika”

The major su$ace glycoprotein (gp63) of Leishmania major incorporated into the immunostimulating complexes (ISCOMs) was used to protect Balblc mice against experimental infection. Two intraperitoneal vaccinations with low doses of gp63 into ISCOMs (gp63-ISCOMs) induced protective immunity in vaccinated mice as indicated by reduced inflammation and suppressed lesions after experimental challenge. An augmented IgG-specific secretion and a specific switching towards the IgG2a isotype was observed in the serum of vaccinated mice. Gp63-ISCOMs primed spleen cells restimulated in vitro with soluble Leishmania antigen (SLA) or live parasites displayed strong gp63-specific proliferative responses and secreted high levels of interleukin-2, interferon 7 and interleukin-10 but not interleukin-4. No delayed type hypersensitivity response to either SLA or LV39 was detected. These data indicate that gp63-ISCOMs induced a protective immunity in the susceptible Balblc mice against Leishmania challenge, modulating the immune response towards a Thl rather than Th2 type. 0 1998Elsevier Science Ltd. All rights reserved Keywords: ISCOMs: Leishmiu:

gp63

The organisms responsible for the leishmaniasis are protozoan parasites of the genus Leishmania’. About 20 species of Leishmnnia are known to infect man leading to symptoms ranging from simple self-healing skin ulcers due to L. major, to severe life-threatening disease, like visceral leishmaniasis, caused by L. donovani, which together constitute an important public health problem’. Chronic cutaneous leishmaniasis caused by L. major in murine model, entirely correlated with the clinical manifestations of human disease, provided an excellent system on the study of the effectiveness of new vaccines. Protective immunity induced by vaccinations is dependent on the capacity of the vaccine to elicit the appropriate immune response able to control or eliminate the pathogen. E,xperimental infections of mice with L. major cause either healing or non-healing cutaneous lesions depending on the CD4+ T-cell subset that dominates.4. It has been shown that Thl cells produce interleukin-2 (IL-2) and interferon 1 which mediate protective cellular immune (IW), responses, whereas Th2 cells product interleukin-4 (IL-4) interleukin-5 (IL-5) and interleukin-10 (IL-IO), which augment. humoral immune responses, allowing the uncontrolled development of lesions, metastasis of the parasite and eventual death of the infected animal?. “Hellenic Pasteur Institute, 127 Vass. Sofias Avenue, 115 21 Athens, Greece. tAuthor to whom correspondence should be addressed. Tel.: +301 64 30 044 (ext. 276, 222); Fax: +301 64 23 498. (Received 24 September 1997; revised version received 25 November 1997; accepted 25 November 1997)

Studies have provided strong evidence that purified leishmanial antigenic components have resulted in significant protection against experimental infection in the murine model”,“‘. Gp63, a 63 kDa protease, is an abundant parasite surface protein which has been implicated in the process of host recognition, attachment and invasion”-‘4. In addition, gp63 has been investigated as a potent vaccine candidate and even host protective in the murine model of cutaneous leishmaniasis”“‘. Both native gp63 and rgp63 are immunogenic to T cells’7,‘x. Gp63 expressed by an oral Salmonella typhimurium vaccine preferentially induces Thl CD4+ cells”, while gp63 incorporated into liposomes has been reported to protect susceptible mice against cutaneous leishmaniasis”‘. Immunostimulating complexes (ISCOMs), first described by Morein et al. in 1984, are cage-like structures, composed by Quiflaja saponaria triterpenoids, cholesterol, phospholipids and multiple copies of one or several antigensZ’,2’. The unique immunogenicity of ISCOMs is attributed to the multimeric presentation of antigens and to the immunomodulatory properties of the inbuilt Quilfaja triterpenoids”. ISCOMs, efficiently delivering viral antigens into both the cytosolic and endosomal pathways of antigen processing, induced protection to several viruses, e.g. HIV II” SIV”, Epstein-Barr*“,“, influenza2x, measles2” and ’ human papilloma viruses”“. In parallel, many amphipathic parasite proteins incorporated into ISCOM formulation have induced antibody response, cell-mediated immunity resulting in protection in several experimental vaccine studies. Toxoplasma gondii tachyzoite surface antigens incorporated into ISCOMs have been studied in sheep and mice”‘-“4 and ISCOMs containing

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a surface sporozoitc protein of Eimcrirr firlcifiwmis have been proved immunogenic after oral administration”. Furthermore, studies on the efticacy of ISCOMs as adjuvants for malaria vaccines have so far focused on antigens of Plosmodium ,ful~ii~Nrllmii’-“~,Araujo and Morein succeeded in inducing remarkable protection against challenge with the virulent blood form of T~panosomu cruzi using ISCOMs as an antigen delivery system and validating the potentiality of ISCOMs in vaccination protocols against the trypanosomidae LeishmrmiLtJ’. In the prcscnt study, a vaccine consisting of gp63 and an ISCOM formulation was used, in order to protect the susceptible mouse strain Balhic from experimental cutaneous leishmaniasis. The generation of systemic protection against Leishmaniu challenge in respect to I,gG subclass distribution, antigen-specific prodiction are T-cell proliferation and cytokine presented here.

MATERIALS AND METHODS Mice Eight-to-nine-week-old inbred female Balb/c mice were used in this study. The animals were maintained under specific pathogen free conditions, receiving a diet of commercial food pellets and water ad lihitum obtained from our own colony. Parasites The strain MRHOISUI59IP of L. major (LV39) was used in all experiments. Promastigotes were grown at 25”C, to stationary phase, in Dulbecco’s modified Eagle medium (Gibco, Paisley, Scotland) supplemented with 10mM Hepes (Gibco), 5 x lO-‘M 2-Me, 2 mM L-glutamine (Sigma, Germany), 45 mM sodium pyruvate (Gibco), 24 mM N,aHCOj (Gibco), 100 g ml streptomycin and 100 units ml-’ penicillin, 10% fetal calf serum (FCS) (Seromed, Berlin, Germany). Soluble Leishmania antigen (SLA) Stationary phase LV39 promastigotes were washed three times in phosphate buffer saline (PBS, Flow Lab, Meckenheim, Germany), adjusted to 1 x 10” promastiby sonication on ice for 2 min gotes ml ‘, disrupted and centrifuged at 15OOg for 30 min. Total protein concentration determination was carried out using the Micro BCA Protein Assay Kit (Pierce, IL;). The concentration determined was 2.7 mg ml . The antigen was aliquoted and stored at -70°C until use. Gp63 The Leishmania membrane glycoprotein gp63 was purified from LV39 cultures by ion exchange chromatography as described by Bouvier et al.“‘.43.Briefly, gp63 was extracted from washed promastigotes in TBS (IO mM Tris-HCI, 5 mM NaCI, pH 7.5) (Sigma, Germany) containing 2% Triton-114 (Merk Darmstadt, FRG). The detergent phase obtained was diluted 12-fold with 10 mM Tris-HCI containing 2.2 mM lauryldodecylamine N-oxide (Sigma) and loaded on a 5 x50 mm column of Fractogel TSK DEAE-650 (Merk, Darmstadt, FRG) and the bound proteins were

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clutcd at a flow rate of I ml min ‘. Fractions were pooled and applied to a Mono Q quartcrnary amine anion cxchanFc column (Pharmacia. Uppsala, Sweden) at I ml min . Protein concentration was determined by the Micro BCA Protein Assay Reagent Kit. Gp634SCOMs The gp63-ISCOMs wcrc prcparcd as described cIscwhereJ’.JS. Briefly, 0.5 mg gp63 mixed with 2.5 mg triterpcnoids, 0.25 mg cholesterol and 0.25 mg phosphatidylcholinc in the presence of IO /lg MEGAIO (Sigma) in a volume of 1 ml. The mixture was cxtensively dialysed against PBS (Gibco) for 24 h at 21°C and then 24 h at 4°C. Free components wcrc rcmovcd by ultraccntrifugation (200000g for I8 h at 21°C) through a 50-10% sucrose gradient in PBS. The pcllct obtained was adjusted to I mg ml ‘. The gp63-ISCOMs were characterized by electron microscopy and sedimentation coefficient. The triterpenoid content was deteryined by reverse phase HPLC (0.75 mg Quil Aml , 0.025 MEGA-IO ml ‘). Vaccination protocol Three groups of 27 mice wcrc vaccinated twice at 15-day intervals with I /lg gp63-ISCOMs, 1 /cg gp63 or PBS, intraperitoneally (i.p.). Twelve of each group of the experimental animals were bled and killed I5 days after the second vaccination and their spleens were obtained for in vitro assays. The rest were used for protection experiments against challenge infection with LV3Y promastigotes. In addition, a group of nine mice was vaccinated i.p. three times at 15-day intervals with 1 /lg gp63 emulsified in complete Freud’s adjuvant, 1 Ltg gp63 emulsified in incomplete Freud’s adjuvant and I /lg gp63, respectively. Fifteen days after the third the animals were tested for specific vaccination, antibody and lymphoproliferative responses. Challenge infection Two weeks after the second vaccination, a volume of 20 /II PBS containing 2 x 105 late stationary phase LV39 promastigotes was injected subcutaneously (s.c.) into the right footpad of vaccinated or unvaccinated control mice. Lesion progression was monitored by measuring footpad thickness using a dial caliper (Kory Seiky MFG. Co. Ltd, Japan) at weekly intervals for I3 weeks. Results were expressed in mm. Detection of gp63-specific antibodies Serum-specific IgG subclasses were determined by a standard enzyme linked immunosorbent assay (ELISA) which was performed as previouslyX7 described. Briefly, microtitre plates (Nunk, Kamstrup, Denmark) were incubated with 0.5 rig ml ’ gp63 and serial serum dilutions. Horseradish peroxidase-conjugated rat antimouse (IgGl, IgG2a, IgG2b and IgG3) mAbs (Pharmingen, San Diego, CA) were used to detect bound antibodies. The serum antibody titres were presented as log,,, of reciprocals of end point dilutions. Secondary in vitro gp63-specific lymphoproliferative responses Spleens from vaccinated or non-vaccinated normal mice were aseptically excised and used to prepare

ISCOMs vaccine against leishmaniasis:

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Statistical

of the mean values of the two groups was assessed by using a Student’s t test. The probability @) of <0.05 was considered to indicate statistical significance.

RESULTS Estimation of challenge infection To evaluate the in viva immune function resulting from the vaccinations, 15 days after the second vaccina-

tion, we challenged all the experimental groups with 2 x IO5LV30 promastigotes and monitored the inflammation and lesion progression for I3 weeks (Figure I). In the case of inflammation and lesion size taken as indicators of the disease status, our results show gp63-ISCOMs vaccinated mice to be significantly more resistant to Leishnzunia infection than the gp63- and non-vaccinated ones. Significant reduction of primary inflammation as well as secondary lesion was observed in the group vaccinated with gp63-ISCOMs compared

Single spleen-cell suspensions prepared from all groups of mice were cultured with 10 /lg ml ’ SLA, IO” LV39 ml ‘, or medium alone and incubated for 24, 48, 72 or 96 h at 37°C in 5% CO? in a humidified incubator. Culture xupernatants were harvested, aliquoted and stored at -70°C until assayed for specific cytokines. Sandwich enzyme immunoassay (SEIA) was used to detect IFN;‘. IL-4 and IL-10 as previously’7 described. Quantitation was performed using a standard dosedependent curve and the cytokine concentrations detected in the samples are presented as pg ml ‘. The detection limits for the cytokines were 125 mg ml ’ for IFN??, 35 pg ml ’ for IL-4 and 60 pg ml ’ for IL-IO. IL-2 was measured using the CTLL cell line which proliferates specifically in response to IL-2 but not IL-4. CTLL cell line was kindly supplied by Dr C.

to the two other ficant difference

Primary

groups @<0.05). There was no signibetween the control non-vaccinated

mice on the inflammation or lesion development those vaccinated with gp63. IgG subclass

distribution

of gp63-specific

and

antibodies

In order to examine the influence of ISCOMs in the humoral response, gp63-specific antibodies were determined in all groups of unvaccinated control and vaccinated mice (Figure 2). Gp63-ISCOMs vaccinated mice produced, 15 days after the second vaccination, a significantly higher level of total IgG-specific serum antibodies than mice vaccinated with gp63 (p
Leclerk, Unite de Biologic des Regulations Immunitaires, Institute Pasteur, Paris, France. Serial dilutions of supernatants were added to CTLL cells in 0.2 ml volume in 96-well plates. After 36 h cultures were pulsed with 1 /l Ci ml ’ “H-thymidine during the final 18 h. Cells were harvested and ‘H-thymidine incorporahgp63

analysis

Data were expressed as the mean values with the standard deviation indicated. The statistical significance

and determination

-R-PBS

et al.

tion was assessed by liquid scintillation counting. IL-2 detection is expressed as Acpm.

single-cell suspensiom (1 x 10” cells ml ‘) in RPMI1640 (Seromcd) culture medium supplemented with IO mM Hepes, 5 x IO ’ M 2-Me, 2 mM L-glutamine, 24 mM NaHC03, 100 units ml ’ penicillin, 100 /~g ml ’ streptomycin and 5% heat inactivated FCS. Cell viability was > 9.5% as determined by trypan blue exclusion. Cells wcrc cultured in round bottom 96-well microtitre plates (Costar, vmbridge, MA) with IO /lg ml ’ SLA, 10” LV3Y ml , or PBS. Cultures were incubated in 50/r, CO, at 37°C for 96 h and pulsed with 1 /ICi ml ’ ‘H-thymidine (Amersam, Co., UK) during the final I8 h. Cells wcrc harvested and ‘H-thymidine incorporation was assessed by liquid scintillation counting. All cultures were performed in triplicate and results are expressed as Acpm.

Cytokine

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+gp63-iscoms Secondary

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Figure 1 Balb/c mice were infected with 2 x lo5 LV39 live promastigotes in the right hind footpad. The primary inflammation and secondary lesion scores are shown as a function of time in mice either unvaccinated (I I) or vaccinated i.p. twice with 1 119 gp63 (v) or 1 /(g gp634SCOMs (1t). The results shown are the mean number +S.D. of footpad swelling and lesion size from 15 individual mice. SD. values < 5% are not shown. Significant differences from the values obtained from the group of mice vaccinated with gp63 indicated with p < 0.05

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0 PBS EI gp63 E gp63-FA

n gp63lscoms

IgG2b

lgG2a

lgG1

lgG3

Figure 2 IgG subclass distribution of gp63-specific antibodies in the serum of Balbic mice either unvaccinated (open bars) or vaccinated i.e. twice with 1 119 gp63 (gray bars), 1 119 gp63-FA (hatched bars) or 1 1’9 gp63-ISCOMs (black bars). The horizontal line indicates the background level. The results given are the mean number *SD. of log,, of serum Ab titre from 15 individual mice. S.D. values ~5% are not shown. Significant differences from the values obtained from the group of mice immunized with gp63 indicated with p < 0.05

primed mice 15 days after the second vaccination. Particularly, live LV39 promastigotes ranging IO’- IO” exhibited higher proliferative responses than SLA. In contrast, spleen cells from mice vaccinated with gp63 showed a moderate proliferation in the presence of either form of antigen, while no proliferation was observed in spleen cells from unvaccinated control mice.

Gp63-ISCOMs resulted in a characteristic IgG subclass distribution of IgG2a > IgG2b > IgGI, while specific antibodies of IgG3 isotypc were close to the background level. In contrast, vaccination of mice with gp63 emulsified in the common FA induced mainly specific antibodies of IgGI isotype.

Gp63-specific

lymphoproliferative responses the possibility whether investigated We gp63-ISCOMs priming T cells could be detected by in v&o restimulation of proliferative activity with either soluble gp63 in the form of SLA or membrane bound in the form of live LV39 promastigotes (Figure 3). Indeed, strong gp63-specific proliferative responses were observed in spleen cells from gp63-ISCOMs

Cytokine production in vitro To examine whether the immune function we observed in vivo was reflected by the cytokine production, WC measured the antigen-specific cytokine pattern induced by gp63-ISCOMs primed spleen cells restimulated in vilru with SLA or LV39. First, the maximal q SLA WLV39

PBS

gp63-FA

gp63

gp63-iscoms

Vaccinations Figure 3 Gp63-specific lymphoproliferative responses of Balb/c mice spleen cells, either unvaccinated, or vaccinated with 1 119 gp63, 1 jrg gp63-FA or 1 119 gp63-ISCOMs restimulated in vitro with 10 119ml ’ SLA (open bars) and lo5 ml ’ LV39 (black bars) promastigotes. The bars represent the ‘H-thymidine incorporation, expressed as Acpm + S.D. from triplicate cultures of pooled spleen cells (n = 3) from two independent experiments. Significant differences from the group of mice vaccinated with gp63 are indicated by p < 0.05

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/SCOMs vaccine secretions of IL-2, IFN]‘, IL-IO and IL-4 were examined regarding dlifferent culture periods (24, 48, 72 and Y6 h). The highest levels of IL-2 secreted at 24 h of the culture period, while IFN],, IL-IO and IL-4 peaked at 72 h of culture period (data not shown). Spleen cells from mice vaccinated either with gp63 or gp63-ISCOMs produced increased lcvcls uf IL-2 upon restimulation in r&~> with both forms of antigen (Figcwc~ 41). Low spontaneous rclcase of IL-2 occurred in cultures of gp63-ISCOMs primed spleen cells, while no IL-2 production was observed in cell cultures obtained from unvaccinated control mice in the presence of antigen. Gp63-ISCOMs immune cells when co-cultured in ritru with LV3c) produced signiticant amounts of both

againstfeishmaniasis: G. Papadopoulou et al.

IFNJ~ and IL-IO, while when co-cultured with SLA they produce lower amounts of the same cytokines (F@tre 411,111). In the same supcrnatants low spontaneous release of IL-IO was also observed. On the contrary, spleen cells from mice vaccinated with gp63 secreted negligible amounts of IFNl* and failed to produce IL-IO. There was net detection uf IFNy or IL-10 in the culture supernatants of unvaccinated control mice with or without antigen stimulation. Spleen cells from mice vaccinated with gp63 when restimulated in vitro either with SLA or LV39 produced enhanced quantities of IL-4 (Figure 40’). Some IL-4 was also observed when gp63-ISCOMs primed spleen cells were cultured with both forms of antigen, but this quantity was significantly less than that

Onil EISLA n LV39

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Figure 4 Cytokine production detected in supernatants of spleen-cell cultures from Balb/c mice either untreated (open bars) or vaccinated with 1 lrg gp63 (gray bars) or 1 /(g gp63-ISCOMs (black bars), after in vitro restimulation with 10 ,rg ml ’ SIA or lo5 ml ’ LV39 promastigotes. IL-Z, IFN;,, IL-10 and IL-4 values are shown as the mean values i S.D. of the values obtained from triplicate cultures of pooled spleen cells (r~= 3) in two independent experiments at the time point of maximal secretion for each cytokine. SD. values < 5% are not shown. Significant differences from the values obtained from the group of mice immunized with gp63 indicated with p co.05

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produced by gp63 primed cells. Spleen cells from unvaccinated control mice did not produce any IL-4 in response to restimulation with antigen ;fr ritro.

DISCUSSION In the present study we have shown that two vaccinations with gp63-ISCOMs induced protective immunity in Balbic mice ‘innately susceptible’ to L. mujor infcction as demonstrated by reduced inflammation and suppressed lesions. In order to analyse the immune functions leeding to the modulation of the susceptible towards the resistant disease phenotype, IgG-specific subclass distribution, antigen-specitic T-cell prolifcration and cytokine production were determined. Fractionated soluble leishmanial antigens either with Curynehncterium parvum or entrapped within liposomes have been used as candidate vaccines against Leishmania infection and these efforts either induced or failed to induce protective antiparasitic immunity”‘.Jb-JX. On the other hand, mice vaccinated with a small number of parasites mounted an acquired resistance to a larger, normally pathogenic challenge, characterized by prolonged delayed type hypersensitivity response (DTH) response, small quantities of IL-4, decreased parasite-specific IgGl antibodies, and increased levels of IFN1’ and IgG2a antibodies14.5”. In our experimental model, vaccination with gp63 into ISCOMs formulation augmented the IgG-specific secretion and a specific switching towards the IgG2a isotype was observed in the serum of Balbic mice. Such a change in isotype production is elucidated by the increased IFNls secretion and the abrogation of IL-4 observed in supernatants of spleen cells primed with gp63-ISCOMs. Moreover, it is known that IFNy. produced by Thl-like cells, both suppresses the induction of IgGl and enhances the production of IgG2a antibody subtype”, and similar types of protection have been reported for many parasite infectiorP3. The systemic antibody production generated by gp63-ISCOMs in mice presupposes the introduction of gp63-epitops into the MHC class II pathway by endocytoses and the activation of CD4+ T cells. Vaccination of Balbic mice with gp63-ISCOMs produced strong antigen-specific proliferative responses in the spleen, confirming previous reports of the ability of ISCOMs to prime for recall proliferative responses in vitro after parenteral immunization’4.55. The strong priming of T cells by gp63-ISCOMs was also confirmed by the antigen-specific IL-2 production which occurred when gp63-ISCOMs primed cells were restimulated with SLA or LV31) in vitro. Moreover, the cytokine pattern observed in gp63-ISCOMs primed T cells shows an also significant production of IFNll, contrary to the production occurring in cultures of gp63 primed cells. Although the ability of ISCOMs and other adjuvants to induce a cytokine response has not been fully claritied”“~57, previous reports have documented that many ISCOMs formulations stimulate the secretion of IL-2 and IFN], in viw %w’ and in vitro”“, and that during immunization saponin increases the expression of class II molecules on antigen presenting cells”‘. Additionally, vaccination with proteins incorporated into ISCOM formulations which promote protein translocation into the cytosol

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et al. succeeds in gcncrating CDX+ CTL in \*itw”‘.“‘. Since the sccrction of IL-2 and IFN?’ detected in our cxpcrimcnts can be due to CD4+ and/or CDs+ T cells which are efticient to produce both cytokines upon stimulation”‘.“‘, further cxpcriments on selectively depleted T-ccl1 subsets arc rcquircd to elucidate the cellular source of this production. The abrogation of antigen-specitic IL-4 production observed in cultures of gp63-ISCOMs primed cells. compared to IL-4 produced by gp63 primed cells. supports the suggestion that a strong Thl-type activation was occurred. This finding was confirmed by previous results whcrc an ISCOM formulation of semidetincd tritcrpenoid compositions carrying inllucnza virus envelope antigens and ISCOMs formulation of highly purified Quilkrjicr fractions containing a recombinant Epstein-Barr virus envclopc protein were also found to stimulate Th I -type rcsponses’s.““.““. A similar Thl type of protective immune response was observed in recent studies on nucleic acid vaccination with a plasmid containing cDNA encoding the gp63”‘. In parallel, the detection of IL-IO in the cultures of spleen cells originating from Balbic mice vaccinated with gp63-ISCOMs indicated that Th2 CD4+ cells have been also activated by gp63-ISCOMs. It is known that IL-IO diminishes the biologic consequences of Thl-cell activation via antigen presenting cells. This is attributed to the down-regulation of the expression of class II MHC molecules or the suppression of the production of parasiticidal metabolites involved in the parasite destruction and various inflammatory mediators”H. We have previously reported that Epstein-Barr virus gp340 envelope protein included in highly purified Quillajia fractions completely abrogates the IL-IO production, indicating that immune responses generated by ISCOMs can be manipulated by altering the triterpenoid composition”. Surprisingly, no DTH response to SLA or live LV3Y promastigotes was detected in gp63-ISCOMs vaccinated mice, although Thl cells are known to induce this hypersensitivity”“. Likewise, oral vaccination against Leishmaniu preferentially induced Th I subsets of CD4+ cells but did not result in a DTH response’“. Nevertheless, earlier studies demonstrated that mice protectively immunized with i.v. doses of killed promastigotes not only failed to develop DTH but spleen cells from these mice could specifically inhibit DTH to leishmanial antigen”‘. Additionally, successful oral immunization of mice with Salmonella typhimurium vaccine expressing gp63 was not followed by DTH development either. These findings suggest the notion that CD4+ T cells may assume heterogeneity beyond the current Thl and Th2 classification”.‘-. In summary, we suggest that gp63 incorporated into ISCOMs formulation can modulate the immune response of vaccinated Balb/c mice preferentially towards a Thl- rather than a Th2-type response, and can induce a partial protection against Lrishmania challenge. The IL-4 abrogation, the enhanced IFN;l production and the IgG2a switching confirm the above suggestion. The Th2-cell activation demonstrated by the IL-IO production could explain the partial protection observed in our experimental system. The capacity of ISCOMs to induce a full range of T-cell responses in viva, the continuous improvement in ISCOMs

lSC0M.s vaccine against leishmaniasis: G. Papadopoulou et al. technology, as well as the selection of gp63 immunogcnic peptides could increase the cfticacy of ISCOMs vaccine so that an exclusive Thl-type response will be accomplished providing full protection in Leishmanicr infections.

ACKNOWLEDGEMENTS The authors thank Dr M. Villacres for her critical assistance in the gp63lSCOMs preparation and Dr B. Sundquist for the triterpcnoid content determination. They also thank Professors B. Morein and S. Haralabidis for a critical review of the manuscript.

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