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A protective cocktail vaccine against murine cutaneous leishmaniasis with DNA encoding cysteine proteinases of Leishmania major
Vaccine 19 (2001) 3369– 3375 www.elsevier.com/locate/vaccine
A protective cocktail vaccine against murine cutaneous leishmaniasis with DNA encoding cysteine proteinases of Leishmania major Sima Rafati a,*, Ali-Hatef Salmanian b,1, Tahere Taheri a, Manije Vafa a, Nicolas Fasel c b
a Department of Immunology, Pasteur Institute of Iran, PO Box 11365 -6699, Tehran, Iran Department of Molecular Biology, Pasteur Institute of Iran, PO Box 11365 -6699 Tehran, Iran c Institute of Biochemistry, Uni6ersity of Lausanne, Epalinges, Switzerland
Received 10 October 2000; received in revised form 31 January 2001; accepted 27 February 2001
Abstract The protection elicited by the intramuscular injection of two plasmid DNAs encoding Leishmania major cysteine proteinase type I (CPb) and type II (CPa) was evaluated in a murine model of experimental cutaneous leishmaniasis. BALB/c mice were immunized either separately or with a cocktail of the two plasmids expressing CPa or CPb. It was only when the cpa and cpb genes were co-injected that long lasting protection against parasite challenge was achieved. Similar protection was also observed when animals were first immunized with cpa/cpb DNA followed by recombinant CPa/CPb boost. Analysis of the immune response showed that protected animals developed a specific Th1 immune response, which was associated with an increase of IFN-g production. This is the first report demonstrating that co-injection of two genes expressing different antigens induces a long lasting protective response, whereas the separate injection of cysteine proteases genes is not protective. © 2001 Published by Elsevier Science Ltd. Keywords: Leishmania; DNA vaccine; Cysteine proteinases
1. Introduction Immunity against Leishmania is dependent on the development of a strong T cell response (mainly T helper 1) involving the production of cytokines such as IFN-g and IL-12 which stimulate the microbicidial activity of macrophages [1,2]. Considerable efforts have been devoted to the development of vaccines against Leishmania infections and crude or purified antigens have been shown to elicit a certain degree of protection in susceptible mouse strains such as BALB/c mice [3,4]. Protection against Leishmania has also been investigated with DNA immunizations [5]. These genetic vaccines are potent activators of innate and adaptive immunity [6,7] as they are able to induce a pro* Corresponding author. Tel.: +98-21-64687614, ext.: 369; fax: +98-21-8742314/5. E-mail address: [email protected] (S. Rafati). 1 Present address: National Center for Genetic Engineering and Biotechnology, Tehran, Iran.
tective immune response in a variety of experimental models. Earlier observations had shown that bacterial DNA possesses immunostimulatory properties, linked to a sequence of dinucleotide CpG, present much more frequently than in bacteria than in vertebrate DNA [8– 10]. Such CpG motifs are considered as a powerful new class of adjuvant that promote CTL, B cell and Th1type responses to proteinaceous antigens [11]. The immunostimulatory property of CpG might be important for redirecting of Th2 response to a protective Th1 response. Thus, CpG oligonucleotides have been shown to trigger a protective and curative response in L. major infected BALB/c mice 15 days after infection [12]. Similarly, susceptible mice injected with a plasmid DNA expressing L. major gp 63 or with LACK DNA are reported to display a significant resistance against infection with the homologous parasite [13,14]. In contrast, injection with the LACK or gp 63 proteins promoted the disease exacerbating Th2 response against L. major infection. A longer lasting protective effect of gp
0264-410X/01/$ - see front matter © 2001 Published by Elsevier Science Ltd. PII: S0264-410X(01)00081-0
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63 gene product was obtained when IL-12 gene was co-injected suggesting that the persistence of IL-12 may be an essential determinant for controlling the infection. Thus, DNA vaccination holds considerable promise for vaccination against diseases in which Th1 responses are desirable. Leishmania and Trypanosomes express large quantities of cysteine proteinases (CP) which are members of the papain superfamily [15]. In Leishmania major, two major CP have been described. CPb is a type I cysteine proteinase present maximally at the amastigote developmental stage [16] and is encoded by a multiple copy gene family with an unusual C-terminal extension. CPa is a type II enzyme first described in L. mexicana. It is expressed at higher level in amastigote stage and in stationary phase promastigote [17]. Recently we isolated the homologous cpa gene from L. major (Accession No. AJ130942, [18]). This gene consists of a single copy, which lacks the long C-terminal extension, and its expression is also specific to the amastigote form of the parasite. We previously reported that native CP isolated from amastigote stage of L. major is recognized by human and murine primed lymphocytes with considerable amount of IFN-g production [19,20]. In this report, we analyzed the induction of a protective immune response in BALB/c mice following genetic vaccination with plasmid DNA encoding CP type I (CPb) and type II (CPa). The protective capacity and the immune response of cpa and cpb plasmids were compared when injected separately or co-injected. In addition, we examined the combination of DNA and protein vaccination of cpa combined with cpb as an alternative way of active immunization.
2. Materials and methods
2.1. Mice and parasites Female BALB/c mice 8– 12 weeks old were obtained from the breeding stock maintained at the Pasteur Institute of Iran. The L. major strain, MRHO/IR/75/ ER was provided by Dr. E. Javadian (School of Public Health, University of Tehran). The parasite were kept in a virulent state by continuous passage in BALB/c mice. Metacyclic infective promastigotes were collected by centrifugation (270× g, 10 min, 4°C), washed three times in PBS (8 mM Na2HPO4, 1.75 mM KH2PO4, 0.25 mM KCl, 137 mM NaCl) and resuspended at a concentration of 2–4×107 cells/ml. This preparation (50 ml) was injected sub-cutaneously in the hind footpad and the course of infection was monitored by weekly measurement of the lesion with a metric caliper. Sizes were compared to the size of the contra-lateral non-infected footpad.
2.2. Plasmid construction and purification The complete open reading frames (without the signal peptide but with the propeptide domain) of cpa (697 bp) and cpb (950 bp) genes were isolated from genomic DNA of L. major [18] and were inserted into the SacI/EcoRI sites of the eukaryotic expression vector pCB6 [21]. This resulted in two constructs, pCB6-cpa and pCB6-cpb, containing the cpa and cpb open reading frames, respectively, under the control of the CMV promoter, inserted downstream of a Kozak consensus sequence and in frame with an initiation codon. Plasmid DNA was purified by polyethylene glycol precipitation (PEG) method [22] which showed the lowest LPS contamination (5 ng/mg) as determined by limulus amebocyte lysate assay (Chromogenix).
2.3. Expression and purification of recombinant cpa and cpb Similarly, the CPa and CPb entire coding segments were inserted into the BamHI/HindIII site of the bacterial expression vector pQE-30 (QIAGEN). Two constructs corresponding to pQE-cpa and pQE-cpb were produced in fusion with an N-terminal histidine (His 6-tag). E. coli M15 pREP4 strain was transformed with pQE-cpa and pQE-cpb and were grown at 37°C with agitation in 500 ml LB supplemented with 100 mg/ml ampicillin and 25 mg/ml kanamycine. The culture was induced with 1 mM isopropyl-1-thio-b-D-galactoside (IPTG) at an OD 600 of 0.8 and grown for a further 4.5 h at 37°C. Cells were pelleted at 4000× g for 10 min, and frozen overnight at −70°C. Bacteria pellets were dissolved in buffer 1 (6M guanidine, 20 mM Tris–HCl, pH 7.9, 500 mM NaCl, 4 mM Octyl b-D-Gluco-pyranoside), incubated for 2 h and then sonicated. After centrifugation (20000× g 10 min, 4°C), the supernatants were passed over a Ni–NTA agarose column (QIAGEN, Chatsworth, CA) and the recombinant CPs were affinity purified via the His 6-tag. The Ni–NTA column was washed with buffer 2 (6 M Urea, 20 mM Tris– HCl, pH 7.9, 500 mM NaCl, 20 mM Imidazole). For removal of urea, the column were washed extensively with buffer 3 (20 mM Tris–HCl, pH 7.9, 150 mM NaCl). Finally, elution of recombinant CPs were obtained by using buffer 4 (20 mM Tris–HCl, pH 4.9, 150 mM NaCl, 100 mM EDTA). The eluted rCPs were concentrated with Amicon and dialyzed against PBS.
2.4. Immunization Five groups of ten female BALB/c mice were injected intramuscularly in thigh skeletal muscle with 100 mg of plasmid DNA in PBS. Group I was injected with pCB6-cpa; group II with pCB6-cpb; group III with pCB6-cpa and pCB6-cpb (100 mg each); group IV with
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pCB6 and group V with PBS. Animals were boosted one month later with the same dose of DNA. Four weeks after the final injection, mice were challenged with 106 stationary phase promastigotes of L. major. Additionally, another group of BALB/c mice (10 females) was injected intramuscularly with pCB6-cpa plus pCB6-cpb as first immunization. For booster injection, they received intraperitoneally a cocktail of rCPa and rCPb (25 mg each) emulsified in IFA. The control group received pCB6 DNA for first injection, followed by intraperitoneal injection of IFA as booster. Both groups were challenged with 106 stationary phase promastigotes of L. major four weeks after last injection.
2.5. T cell proliferation assay and cytokine determination Immediately before challenge and 8 weeks after infection, two mice from each group were killed and the spleen cells were harvested. Two samples of single cell preparations were plated each one in triplicate in 96well microtiter plate at a density of 2×105 cells/well. Ten mg/ml of the respective recombinant antigen (rCPa or rCPb) and 4 mg/well of freeze/thawed L. major were added. Concanavalin A (ConA) was used at a concentration of 8 mg/ml. Three days later, the supernatants were harvested and IFN-g and IL-5 production were assayed by sandwich ELISA. Briefly, the wells of Immunol-1 microtiter plates (Dynatech, Laboratories) were coated with protein G purified rat anti-mouse IFN-g mAb R4-6A2 at 2 mg/ml in PBS by overnight incubation at 4°C. Wells were then washed three times with PBS/0.05% Tween-20 and blocked by incubation with PBS/1% BSA for 2 h at 37°C. The culture supernatants and recombinant IFN-g were then added to individual wells and incubated for overnight at 4°C. The wells were washed three times with PBS/0.05% tween-20 and then biotinylated rat anti-mouse IFN-g mAb AN 18.17.24 was added and incubated for 1 h at 37°C (all cell lines kindly provided by DNAX research Institute Polo Alto, Calif). For the detection of bound biotinylated rat Ab, 100 ml of streptavidine-alkaline phosphatase conjugate (diluted 1/1000) was added to each well for 1 h at 37°C, and following further washing, binding was visualized with O-phenylenediamine (OPD) as substrate. The absorbance was subsequently measured at 492 nm on a titertek multiscan plate reader. IFN-g concentration in the cell cultures were determined from standard culture and concentration from the supernatants higher than minimal values obtained from the standard were considered positive (minimal values is 30 pg/ml). For IL-5, the coating Ab, TRFK4 at 2 mg/ml and biotinylated rat anti-mouse TRFK5 at 1 mg/ml (PharMingen, San Diego, Ca) were used. For standard curves, rIL-5 (PharMingen, San
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Diago, CA) was used. Minimal values considered for IL-5 was 80 pg/ml.
2.6. Measurement of CP-specific antibody responses Pooled serum samples (7–10 mice/group) were obtained at eight weeks after challenge. All samples were stored at −20°C before analysis for specific Ab content. CP-specific IgG1 and IgG2a were measured by ELISA. In brief, 96 well maxisorb plates (NUNC) were coated with rCPa or rCPb (10 mg/ml) overnight at 4°C. Plates were blocked with 1X PBS/1% BSA at 37°C for 2 h to prevent non-specific binding. Sera were added (with dilution of 1:500) and incubated 2 h at 37°C. Biotinylated rabbit anti-mouse IgG1 and IgG2a (all from PharMingen, San Diego, CA) were added for 2 h at 37°C, then incubated (1 h at room temperature) with streptavidine-horseradish peroxidase (BRL, Gaithersburg, MD). Binding of conjugate was visualized with OPD in 0.1 M Sodium Citrate, pH 4.5 containing 0.03% H2O2. The color reaction was stopped by adding 4 N H2SO4, and the absorbance was measured at 492 nm.
2.7. Statistical analysis The difference in the level of protection, cytokine and antibody production was determined by one way ANOVA and the students t-test. Differences were considered significant when PB0.05.
3. Results
3.1. Efficacy of DNA 6accination with plasmid DNAs encoding CPa or CPb or a cocktail of the two plasmids The efficacy of DNA vaccinations with plasmid DNAs encoding CPa or CPb or a cocktail of the two plasmids was evaluated by their capability to induce protection and a specific immune response to Leishmania infection in the BALB/c mice cutaneous model. Footpad swelling of five different groups of mice (10 per group) was measured after a challenge inoculation with 106 highly infective promastigotes of L. major (Fig. 1). All mice which have received the DNA vector without any insert (group IV) or PBS (group V), showed significant lesions by 4 weeks post-challenge. A delay in the growth of the lesions was observed in the animals of group IV compared to the animals of group V which had to be euthanised by week 8. This difference is likely to be due to a specific immunostimulation induced by the CpG motif present on the expression vector DNA pCB6. No difference was observed when animals received 100 mg or 200 mg of empty vector,
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confirming that the level of protection observed in group III was not simply due to a difference in the amount of injected DNA (data not shown). Vaccination with plasmid DNA encoding CPa (group I) or CPb (group II) delayed footpad swelling when compared to immunization with PBS alone (group V). For a certain time, immunization with the cpb gene had a significant effect in delaying footpad swelling. However, this effect was not long lasting and 11 weeks after challenge, the protective effect disappeared and the lesions reached the size of the lesions in the animals of group I and IV. Thus, at week 13 after challenge, no statistically significant differences can be observed between groups I, II and IV. In these groups, the footpad lesions progressed to such an extent that animals had to be euthanised either at week 11 (group I and IV) or week 13 (group II). By contrast, vaccination with a cocktail of cpa and cpb genes (group III) resulted in control of the infection. Footpad swelling ceased and all the animals displayed partial protection 14 weeks after infectious challenge.
3.2. Cocktail DNA 6accination with cpa/cpb genes enhances IFN-k production The production of IFN-g, as measured immediately before and two months after challenge (parasite infection), in response to both Leishmania lysate and recombinant CPs by spleen cells are presented in Fig. 2. Spleen cells from mice of group II and III in which the
infection was controlled at week 8, produced high amounts of IFN-g before (9009 102 and 10009119 pg/ml, respectively) and after infection (30009 125 and 25009 100 pg/ml, respectively) in response to the recombinant CPS and to L. major lysate (60009 284 and 80009 304 pg/ml, respectively). The disease was markedly attenuated and stabilized, up to 14 weeks following vaccination, in group III mice vaccinated with 200 mg of a cocktail of cpa and cpb genes. However, in spite of high production of IFN-g by group II and after an initial delay in footpad swelling, the protective effect disappeared and the animals finally had to be sacrificed by week 14 when footpad swelling had become too severe. On the contrary, the animals which were immunized with cpa gene (group I) alone, showed a lower level of IFN-g production to both rCPa and Leishmania lysate (7009 65 and 20009 120 pg/ml, respectively) after infection and protection was not achieved. No IFN-g production was detected in supernatants of spleen cells of mice in the two control groups of IV and V, in response to recombinant CP and to L. major lysate. No IL-5 was detectable in the supernatant of cells from group II and group III after stimulation with rCPs or Leishmania lysate. In contrast, significant levels of IL-5 were detected in the supernatant of spleen cells from group I (1309 28 pg/ml in response to rCPa and 1569 36 pg/ml to Leishmania lysate), group IV (1709 50 pg/ml in response to Leishmania lysate) and group V (5509109 pg/ml to Leishmania lysate) at eight weeks after infection.
Fig. 1. Representation of footpad swelling and protective immunity following pCB6-cps immunization. BALB/c mice, ten animals in each group, were immunized intramuscularly with pCB6-cpa (group I); pCB6-cpb (group II); pCB6-cpa and pCB6-cpb (group III); pCB6, vector alone (group IV); and PBS (group V) and boosted 4 weeks later. Mice were challenged in their right hind footpads with 106 metacyclic L. major promastigotes four weeks after booster. (**): time of euthanasia.
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Fig. 2. In vitro production of IFN-g by spleen cells of DNA vaccinated mice before infection and at week 8 after challenge with L. major. Each group was restimulated by the corresponding recombinant proteins (10 mg/ml). Individual mice (n =2) were euthanized and the spleens were harvested at week 8 after infection. Single cell preparations were plated in triplicate in 96 well microtitre plates at 2 ×105 cells/well in media alone or with respective recombinant cysteine proteinase. 72 h later, supernatants were harvested and IFN-g was assayed by ELISA. Group I: rCPa, group II: rCPb, group III: rCPa and rCPb. Also spleen cells of each group were stimulated with Leishmania lysate (F/T) (4 mg/well) at week 8 after infection. Production of IFN-g in control groups were B 30 pg/ml. The results are shown as mean 9S.D. of triplicate assay of two mice.
3.3. Immunization of BALB/c mice with a cocktail of cpa and cpb and rCPs It was of additional interest to determine whether the combination of DNA and protein immunization with cpa/cpb could induce protective immunity against infection with L. major. To this end, BALB/c mice were immunized with a cocktail of cpa/cpb genes and boosted with a cocktail of recombinant CPa/CPb in the presence of IFA. As shown in Fig. 3, footpad swelling in vaccinated mice compared to the control group suggested a high level of protective immunity. Interestingly, the level of IFN-g production following the booster injection was more than three fold higher than the first immunization with DNA (Table 1). In contrast to this observation, BALB/c mice immunized with recombinant CPs in presence of IFA were not able to protect mice against infection with L. major (data not shown). These results demonstrated that the protective response of cpa/cpb was only relevant when immunization commenced with DNA and was followed by booster injection with either recombinant CPs or cp genes.
3.4. CPs-specific production of IgG1 and IgG2a correlated with protecti6e effect of DNA 6accination To compare IgG isotype in protective and non protective vaccinated groups, sera were collected 8 weeks after challenge and assessed for IgG1 and IgG2a. As it is shown in Fig. 4, although the highest level of IgG1
production is observed in group I (pCB6-cpa), there was no significant difference observed in all other groups. Mice that developed an effective protective response (e.g., the group which was vaccinated with DNA and protein) had substantially higher levels of CP-specific IgG2a antibody compared with unprotected mice. This is consistent with the results obtained above that a combination of DNA and protein (CPa/CPb) preferentially induced a Th1 response.
4. Discussion Gene vaccination is a promising approach to immunize animals and humans against pathogenic microorganisms. In this report, we showed that immunization of genetically susceptible BALB/c mice using cocktail of type I (CPb) and type II (CPa) CP had the capacity to induce long lasting protection against the L. major infection. Analysis of the immune response showed that protected animals developed a specific Th1 immune response to Leishmania lysate. It is likely that the enhanced production of IFN-g before infection either in group III or in the group which received the combination of DNA and recombinant proteins, provides the milieu for the development of protective Th1 response. The resistance developed against L. major infection in these groups could be attributed to the expression of the two cp genes. These results corroborate previously reported data [14] demonstrating that vaccination with plasmid DNA encoding a specific leishmanial antigen is
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Fig. 3. Evaluation of protection against infection with L. major, as represented by footpad swelling in BALB/c mice immunized with a mixture of cpa/cpb genes and boosted with a cocktail rCPa/rCPb in the presence of IFA. The control group received the cloning vector (pCB6) as first injection and four weeks later was injected with IFA. Both groups were challenged with 106 metacyclic parasites four weeks after the booster.
more effective than immunization with the leishmanial protein combined with recombinant IL-12 in eliciting long-term immunity which is capable of controlling L. major infection. Immunostimulatory DNA sequences are likely to exert systemic effects via an IL-12 and IFN-g-dependent mechanism [23,24]. The apparent advantage of DNA vaccination is due to the intracellular antigen production and the adjuvant effect of bacterial plasmid DNA [24]. Our work provides strong evidence that DNA vaccination with a cocktail of two cp genes elicits a Th1 response as demonstrated by IFNg production and Ig subclass typing and also induces resistance in BALB/c mice. Therefore, these results provide a basis for further studies towards the use of cocktail DNA vaccines in protection studies against L. major infection. The data presented here indicate that the protective response of CPa/CPb is achieved only when immunization is commenced with plasmid DNAs and then followed by booster injection with either recombinant CPs or cp genes. Thus the DNA encoded CPa/CPb have the capability of inducing Th1-type cell mediated responses and have influences on early decisions of the immune system. The CP proteins may have immunological properties of CPs that are involved in modulating the hosts immune responses as reported for parasite survival and proliferation in L. mexicana mutants lacking cysteine proteinase genes such as cpa, cpb or both cpa and cpb [25]. It has been shown that L. mexicana CP
is a T cell immunogen that could induce protective Th1 cell line [26]. Similarly, L. amazonesis CP provided some protection against subsequent challenge through the induction a Th1-associated response [27]. This is the first report demonstrating that co-injection of two genes expressing different antigens induces a protective response, whereas the separate injection of cp genes is not protective. In addition, immunization of BALB/c mice with combination of DNA and protein immunization can be as effective as DNA immunization which present an alternative way of immunization and vaccine development in the prevention of leishmaniasis.
Table 1 IFN-g production following cps DNA vaccination as 1st immunization and recombinant CPs (rCPa and rCPb) as booster injection in BALB/c mice Immunization time
4 weeks after first immunization 4 weeks after second immunization 8 weeks after challenge
IFN-g (pg/ml) production in response to rCPa+rCPb
Leishmania lysate
1300 9 285
800 9176
4800 9 405
1400 9224
3100 9320
2000 9398
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[10] [11]
[12]
[13]
Fig. 4. IgG1 and IgG2a profile of CP-specific antibodies induced by immunization with cps DNA. Mice were bled at week 8 after challenge and pools of 7 – 10 mice/group were analyzed for presence of CPs-reactive IgG1 and IgG2a.
Acknowledgements We wish to thank Dr J. Mauel, Dr I. Fischberg, Dr Ruddger and Dr S. Masina for their helpful advice and revision of the paper. This investigation received financial support from UNDP/World Bank/WHO Special, Program for Research and Training in Tropical Disease (TDR), ID c970556. This work was also supported by grants nos. 31-47342.96 and 31-59450.99 from the FNRS to N.F who was the recipient of the Dr Max Cloetta fellowship (Switzerland).
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A protective cocktail vaccine against murine cutaneous leishmaniasis with DNA encoding cysteine proteinases of Leishmania major Sima Rafati a,*, Ali-Hatef Salmanian b,1, Tahere Taheri a, Manije Vafa a, Nicolas Fasel c b
a Department of Immunology, Pasteur Institute of Iran, PO Box 11365 -6699, Tehran, Iran Department of Molecular Biology, Pasteur Institute of Iran, PO Box 11365 -6699 Tehran, Iran c Institute of Biochemistry, Uni6ersity of Lausanne, Epalinges, Switzerland
Received 10 October 2000; received in revised form 31 January 2001; accepted 27 February 2001
Abstract The protection elicited by the intramuscular injection of two plasmid DNAs encoding Leishmania major cysteine proteinase type I (CPb) and type II (CPa) was evaluated in a murine model of experimental cutaneous leishmaniasis. BALB/c mice were immunized either separately or with a cocktail of the two plasmids expressing CPa or CPb. It was only when the cpa and cpb genes were co-injected that long lasting protection against parasite challenge was achieved. Similar protection was also observed when animals were first immunized with cpa/cpb DNA followed by recombinant CPa/CPb boost. Analysis of the immune response showed that protected animals developed a specific Th1 immune response, which was associated with an increase of IFN-g production. This is the first report demonstrating that co-injection of two genes expressing different antigens induces a long lasting protective response, whereas the separate injection of cysteine proteases genes is not protective. © 2001 Published by Elsevier Science Ltd. Keywords: Leishmania; DNA vaccine; Cysteine proteinases
1. Introduction Immunity against Leishmania is dependent on the development of a strong T cell response (mainly T helper 1) involving the production of cytokines such as IFN-g and IL-12 which stimulate the microbicidial activity of macrophages [1,2]. Considerable efforts have been devoted to the development of vaccines against Leishmania infections and crude or purified antigens have been shown to elicit a certain degree of protection in susceptible mouse strains such as BALB/c mice [3,4]. Protection against Leishmania has also been investigated with DNA immunizations [5]. These genetic vaccines are potent activators of innate and adaptive immunity [6,7] as they are able to induce a pro* Corresponding author. Tel.: +98-21-64687614, ext.: 369; fax: +98-21-8742314/5. E-mail address: [email protected] (S. Rafati). 1 Present address: National Center for Genetic Engineering and Biotechnology, Tehran, Iran.
tective immune response in a variety of experimental models. Earlier observations had shown that bacterial DNA possesses immunostimulatory properties, linked to a sequence of dinucleotide CpG, present much more frequently than in bacteria than in vertebrate DNA [8– 10]. Such CpG motifs are considered as a powerful new class of adjuvant that promote CTL, B cell and Th1type responses to proteinaceous antigens [11]. The immunostimulatory property of CpG might be important for redirecting of Th2 response to a protective Th1 response. Thus, CpG oligonucleotides have been shown to trigger a protective and curative response in L. major infected BALB/c mice 15 days after infection [12]. Similarly, susceptible mice injected with a plasmid DNA expressing L. major gp 63 or with LACK DNA are reported to display a significant resistance against infection with the homologous parasite [13,14]. In contrast, injection with the LACK or gp 63 proteins promoted the disease exacerbating Th2 response against L. major infection. A longer lasting protective effect of gp
0264-410X/01/$ - see front matter © 2001 Published by Elsevier Science Ltd. PII: S0264-410X(01)00081-0
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63 gene product was obtained when IL-12 gene was co-injected suggesting that the persistence of IL-12 may be an essential determinant for controlling the infection. Thus, DNA vaccination holds considerable promise for vaccination against diseases in which Th1 responses are desirable. Leishmania and Trypanosomes express large quantities of cysteine proteinases (CP) which are members of the papain superfamily [15]. In Leishmania major, two major CP have been described. CPb is a type I cysteine proteinase present maximally at the amastigote developmental stage [16] and is encoded by a multiple copy gene family with an unusual C-terminal extension. CPa is a type II enzyme first described in L. mexicana. It is expressed at higher level in amastigote stage and in stationary phase promastigote [17]. Recently we isolated the homologous cpa gene from L. major (Accession No. AJ130942, [18]). This gene consists of a single copy, which lacks the long C-terminal extension, and its expression is also specific to the amastigote form of the parasite. We previously reported that native CP isolated from amastigote stage of L. major is recognized by human and murine primed lymphocytes with considerable amount of IFN-g production [19,20]. In this report, we analyzed the induction of a protective immune response in BALB/c mice following genetic vaccination with plasmid DNA encoding CP type I (CPb) and type II (CPa). The protective capacity and the immune response of cpa and cpb plasmids were compared when injected separately or co-injected. In addition, we examined the combination of DNA and protein vaccination of cpa combined with cpb as an alternative way of active immunization.
2. Materials and methods
2.1. Mice and parasites Female BALB/c mice 8– 12 weeks old were obtained from the breeding stock maintained at the Pasteur Institute of Iran. The L. major strain, MRHO/IR/75/ ER was provided by Dr. E. Javadian (School of Public Health, University of Tehran). The parasite were kept in a virulent state by continuous passage in BALB/c mice. Metacyclic infective promastigotes were collected by centrifugation (270× g, 10 min, 4°C), washed three times in PBS (8 mM Na2HPO4, 1.75 mM KH2PO4, 0.25 mM KCl, 137 mM NaCl) and resuspended at a concentration of 2–4×107 cells/ml. This preparation (50 ml) was injected sub-cutaneously in the hind footpad and the course of infection was monitored by weekly measurement of the lesion with a metric caliper. Sizes were compared to the size of the contra-lateral non-infected footpad.
2.2. Plasmid construction and purification The complete open reading frames (without the signal peptide but with the propeptide domain) of cpa (697 bp) and cpb (950 bp) genes were isolated from genomic DNA of L. major [18] and were inserted into the SacI/EcoRI sites of the eukaryotic expression vector pCB6 [21]. This resulted in two constructs, pCB6-cpa and pCB6-cpb, containing the cpa and cpb open reading frames, respectively, under the control of the CMV promoter, inserted downstream of a Kozak consensus sequence and in frame with an initiation codon. Plasmid DNA was purified by polyethylene glycol precipitation (PEG) method [22] which showed the lowest LPS contamination (5 ng/mg) as determined by limulus amebocyte lysate assay (Chromogenix).
2.3. Expression and purification of recombinant cpa and cpb Similarly, the CPa and CPb entire coding segments were inserted into the BamHI/HindIII site of the bacterial expression vector pQE-30 (QIAGEN). Two constructs corresponding to pQE-cpa and pQE-cpb were produced in fusion with an N-terminal histidine (His 6-tag). E. coli M15 pREP4 strain was transformed with pQE-cpa and pQE-cpb and were grown at 37°C with agitation in 500 ml LB supplemented with 100 mg/ml ampicillin and 25 mg/ml kanamycine. The culture was induced with 1 mM isopropyl-1-thio-b-D-galactoside (IPTG) at an OD 600 of 0.8 and grown for a further 4.5 h at 37°C. Cells were pelleted at 4000× g for 10 min, and frozen overnight at −70°C. Bacteria pellets were dissolved in buffer 1 (6M guanidine, 20 mM Tris–HCl, pH 7.9, 500 mM NaCl, 4 mM Octyl b-D-Gluco-pyranoside), incubated for 2 h and then sonicated. After centrifugation (20000× g 10 min, 4°C), the supernatants were passed over a Ni–NTA agarose column (QIAGEN, Chatsworth, CA) and the recombinant CPs were affinity purified via the His 6-tag. The Ni–NTA column was washed with buffer 2 (6 M Urea, 20 mM Tris– HCl, pH 7.9, 500 mM NaCl, 20 mM Imidazole). For removal of urea, the column were washed extensively with buffer 3 (20 mM Tris–HCl, pH 7.9, 150 mM NaCl). Finally, elution of recombinant CPs were obtained by using buffer 4 (20 mM Tris–HCl, pH 4.9, 150 mM NaCl, 100 mM EDTA). The eluted rCPs were concentrated with Amicon and dialyzed against PBS.
2.4. Immunization Five groups of ten female BALB/c mice were injected intramuscularly in thigh skeletal muscle with 100 mg of plasmid DNA in PBS. Group I was injected with pCB6-cpa; group II with pCB6-cpb; group III with pCB6-cpa and pCB6-cpb (100 mg each); group IV with
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pCB6 and group V with PBS. Animals were boosted one month later with the same dose of DNA. Four weeks after the final injection, mice were challenged with 106 stationary phase promastigotes of L. major. Additionally, another group of BALB/c mice (10 females) was injected intramuscularly with pCB6-cpa plus pCB6-cpb as first immunization. For booster injection, they received intraperitoneally a cocktail of rCPa and rCPb (25 mg each) emulsified in IFA. The control group received pCB6 DNA for first injection, followed by intraperitoneal injection of IFA as booster. Both groups were challenged with 106 stationary phase promastigotes of L. major four weeks after last injection.
2.5. T cell proliferation assay and cytokine determination Immediately before challenge and 8 weeks after infection, two mice from each group were killed and the spleen cells were harvested. Two samples of single cell preparations were plated each one in triplicate in 96well microtiter plate at a density of 2×105 cells/well. Ten mg/ml of the respective recombinant antigen (rCPa or rCPb) and 4 mg/well of freeze/thawed L. major were added. Concanavalin A (ConA) was used at a concentration of 8 mg/ml. Three days later, the supernatants were harvested and IFN-g and IL-5 production were assayed by sandwich ELISA. Briefly, the wells of Immunol-1 microtiter plates (Dynatech, Laboratories) were coated with protein G purified rat anti-mouse IFN-g mAb R4-6A2 at 2 mg/ml in PBS by overnight incubation at 4°C. Wells were then washed three times with PBS/0.05% Tween-20 and blocked by incubation with PBS/1% BSA for 2 h at 37°C. The culture supernatants and recombinant IFN-g were then added to individual wells and incubated for overnight at 4°C. The wells were washed three times with PBS/0.05% tween-20 and then biotinylated rat anti-mouse IFN-g mAb AN 18.17.24 was added and incubated for 1 h at 37°C (all cell lines kindly provided by DNAX research Institute Polo Alto, Calif). For the detection of bound biotinylated rat Ab, 100 ml of streptavidine-alkaline phosphatase conjugate (diluted 1/1000) was added to each well for 1 h at 37°C, and following further washing, binding was visualized with O-phenylenediamine (OPD) as substrate. The absorbance was subsequently measured at 492 nm on a titertek multiscan plate reader. IFN-g concentration in the cell cultures were determined from standard culture and concentration from the supernatants higher than minimal values obtained from the standard were considered positive (minimal values is 30 pg/ml). For IL-5, the coating Ab, TRFK4 at 2 mg/ml and biotinylated rat anti-mouse TRFK5 at 1 mg/ml (PharMingen, San Diego, Ca) were used. For standard curves, rIL-5 (PharMingen, San
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Diago, CA) was used. Minimal values considered for IL-5 was 80 pg/ml.
2.6. Measurement of CP-specific antibody responses Pooled serum samples (7–10 mice/group) were obtained at eight weeks after challenge. All samples were stored at −20°C before analysis for specific Ab content. CP-specific IgG1 and IgG2a were measured by ELISA. In brief, 96 well maxisorb plates (NUNC) were coated with rCPa or rCPb (10 mg/ml) overnight at 4°C. Plates were blocked with 1X PBS/1% BSA at 37°C for 2 h to prevent non-specific binding. Sera were added (with dilution of 1:500) and incubated 2 h at 37°C. Biotinylated rabbit anti-mouse IgG1 and IgG2a (all from PharMingen, San Diego, CA) were added for 2 h at 37°C, then incubated (1 h at room temperature) with streptavidine-horseradish peroxidase (BRL, Gaithersburg, MD). Binding of conjugate was visualized with OPD in 0.1 M Sodium Citrate, pH 4.5 containing 0.03% H2O2. The color reaction was stopped by adding 4 N H2SO4, and the absorbance was measured at 492 nm.
2.7. Statistical analysis The difference in the level of protection, cytokine and antibody production was determined by one way ANOVA and the students t-test. Differences were considered significant when PB0.05.
3. Results
3.1. Efficacy of DNA 6accination with plasmid DNAs encoding CPa or CPb or a cocktail of the two plasmids The efficacy of DNA vaccinations with plasmid DNAs encoding CPa or CPb or a cocktail of the two plasmids was evaluated by their capability to induce protection and a specific immune response to Leishmania infection in the BALB/c mice cutaneous model. Footpad swelling of five different groups of mice (10 per group) was measured after a challenge inoculation with 106 highly infective promastigotes of L. major (Fig. 1). All mice which have received the DNA vector without any insert (group IV) or PBS (group V), showed significant lesions by 4 weeks post-challenge. A delay in the growth of the lesions was observed in the animals of group IV compared to the animals of group V which had to be euthanised by week 8. This difference is likely to be due to a specific immunostimulation induced by the CpG motif present on the expression vector DNA pCB6. No difference was observed when animals received 100 mg or 200 mg of empty vector,
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confirming that the level of protection observed in group III was not simply due to a difference in the amount of injected DNA (data not shown). Vaccination with plasmid DNA encoding CPa (group I) or CPb (group II) delayed footpad swelling when compared to immunization with PBS alone (group V). For a certain time, immunization with the cpb gene had a significant effect in delaying footpad swelling. However, this effect was not long lasting and 11 weeks after challenge, the protective effect disappeared and the lesions reached the size of the lesions in the animals of group I and IV. Thus, at week 13 after challenge, no statistically significant differences can be observed between groups I, II and IV. In these groups, the footpad lesions progressed to such an extent that animals had to be euthanised either at week 11 (group I and IV) or week 13 (group II). By contrast, vaccination with a cocktail of cpa and cpb genes (group III) resulted in control of the infection. Footpad swelling ceased and all the animals displayed partial protection 14 weeks after infectious challenge.
3.2. Cocktail DNA 6accination with cpa/cpb genes enhances IFN-k production The production of IFN-g, as measured immediately before and two months after challenge (parasite infection), in response to both Leishmania lysate and recombinant CPs by spleen cells are presented in Fig. 2. Spleen cells from mice of group II and III in which the
infection was controlled at week 8, produced high amounts of IFN-g before (9009 102 and 10009119 pg/ml, respectively) and after infection (30009 125 and 25009 100 pg/ml, respectively) in response to the recombinant CPS and to L. major lysate (60009 284 and 80009 304 pg/ml, respectively). The disease was markedly attenuated and stabilized, up to 14 weeks following vaccination, in group III mice vaccinated with 200 mg of a cocktail of cpa and cpb genes. However, in spite of high production of IFN-g by group II and after an initial delay in footpad swelling, the protective effect disappeared and the animals finally had to be sacrificed by week 14 when footpad swelling had become too severe. On the contrary, the animals which were immunized with cpa gene (group I) alone, showed a lower level of IFN-g production to both rCPa and Leishmania lysate (7009 65 and 20009 120 pg/ml, respectively) after infection and protection was not achieved. No IFN-g production was detected in supernatants of spleen cells of mice in the two control groups of IV and V, in response to recombinant CP and to L. major lysate. No IL-5 was detectable in the supernatant of cells from group II and group III after stimulation with rCPs or Leishmania lysate. In contrast, significant levels of IL-5 were detected in the supernatant of spleen cells from group I (1309 28 pg/ml in response to rCPa and 1569 36 pg/ml to Leishmania lysate), group IV (1709 50 pg/ml in response to Leishmania lysate) and group V (5509109 pg/ml to Leishmania lysate) at eight weeks after infection.
Fig. 1. Representation of footpad swelling and protective immunity following pCB6-cps immunization. BALB/c mice, ten animals in each group, were immunized intramuscularly with pCB6-cpa (group I); pCB6-cpb (group II); pCB6-cpa and pCB6-cpb (group III); pCB6, vector alone (group IV); and PBS (group V) and boosted 4 weeks later. Mice were challenged in their right hind footpads with 106 metacyclic L. major promastigotes four weeks after booster. (**): time of euthanasia.
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Fig. 2. In vitro production of IFN-g by spleen cells of DNA vaccinated mice before infection and at week 8 after challenge with L. major. Each group was restimulated by the corresponding recombinant proteins (10 mg/ml). Individual mice (n =2) were euthanized and the spleens were harvested at week 8 after infection. Single cell preparations were plated in triplicate in 96 well microtitre plates at 2 ×105 cells/well in media alone or with respective recombinant cysteine proteinase. 72 h later, supernatants were harvested and IFN-g was assayed by ELISA. Group I: rCPa, group II: rCPb, group III: rCPa and rCPb. Also spleen cells of each group were stimulated with Leishmania lysate (F/T) (4 mg/well) at week 8 after infection. Production of IFN-g in control groups were B 30 pg/ml. The results are shown as mean 9S.D. of triplicate assay of two mice.
3.3. Immunization of BALB/c mice with a cocktail of cpa and cpb and rCPs It was of additional interest to determine whether the combination of DNA and protein immunization with cpa/cpb could induce protective immunity against infection with L. major. To this end, BALB/c mice were immunized with a cocktail of cpa/cpb genes and boosted with a cocktail of recombinant CPa/CPb in the presence of IFA. As shown in Fig. 3, footpad swelling in vaccinated mice compared to the control group suggested a high level of protective immunity. Interestingly, the level of IFN-g production following the booster injection was more than three fold higher than the first immunization with DNA (Table 1). In contrast to this observation, BALB/c mice immunized with recombinant CPs in presence of IFA were not able to protect mice against infection with L. major (data not shown). These results demonstrated that the protective response of cpa/cpb was only relevant when immunization commenced with DNA and was followed by booster injection with either recombinant CPs or cp genes.
3.4. CPs-specific production of IgG1 and IgG2a correlated with protecti6e effect of DNA 6accination To compare IgG isotype in protective and non protective vaccinated groups, sera were collected 8 weeks after challenge and assessed for IgG1 and IgG2a. As it is shown in Fig. 4, although the highest level of IgG1
production is observed in group I (pCB6-cpa), there was no significant difference observed in all other groups. Mice that developed an effective protective response (e.g., the group which was vaccinated with DNA and protein) had substantially higher levels of CP-specific IgG2a antibody compared with unprotected mice. This is consistent with the results obtained above that a combination of DNA and protein (CPa/CPb) preferentially induced a Th1 response.
4. Discussion Gene vaccination is a promising approach to immunize animals and humans against pathogenic microorganisms. In this report, we showed that immunization of genetically susceptible BALB/c mice using cocktail of type I (CPb) and type II (CPa) CP had the capacity to induce long lasting protection against the L. major infection. Analysis of the immune response showed that protected animals developed a specific Th1 immune response to Leishmania lysate. It is likely that the enhanced production of IFN-g before infection either in group III or in the group which received the combination of DNA and recombinant proteins, provides the milieu for the development of protective Th1 response. The resistance developed against L. major infection in these groups could be attributed to the expression of the two cp genes. These results corroborate previously reported data [14] demonstrating that vaccination with plasmid DNA encoding a specific leishmanial antigen is
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Fig. 3. Evaluation of protection against infection with L. major, as represented by footpad swelling in BALB/c mice immunized with a mixture of cpa/cpb genes and boosted with a cocktail rCPa/rCPb in the presence of IFA. The control group received the cloning vector (pCB6) as first injection and four weeks later was injected with IFA. Both groups were challenged with 106 metacyclic parasites four weeks after the booster.
more effective than immunization with the leishmanial protein combined with recombinant IL-12 in eliciting long-term immunity which is capable of controlling L. major infection. Immunostimulatory DNA sequences are likely to exert systemic effects via an IL-12 and IFN-g-dependent mechanism [23,24]. The apparent advantage of DNA vaccination is due to the intracellular antigen production and the adjuvant effect of bacterial plasmid DNA [24]. Our work provides strong evidence that DNA vaccination with a cocktail of two cp genes elicits a Th1 response as demonstrated by IFNg production and Ig subclass typing and also induces resistance in BALB/c mice. Therefore, these results provide a basis for further studies towards the use of cocktail DNA vaccines in protection studies against L. major infection. The data presented here indicate that the protective response of CPa/CPb is achieved only when immunization is commenced with plasmid DNAs and then followed by booster injection with either recombinant CPs or cp genes. Thus the DNA encoded CPa/CPb have the capability of inducing Th1-type cell mediated responses and have influences on early decisions of the immune system. The CP proteins may have immunological properties of CPs that are involved in modulating the hosts immune responses as reported for parasite survival and proliferation in L. mexicana mutants lacking cysteine proteinase genes such as cpa, cpb or both cpa and cpb [25]. It has been shown that L. mexicana CP
is a T cell immunogen that could induce protective Th1 cell line [26]. Similarly, L. amazonesis CP provided some protection against subsequent challenge through the induction a Th1-associated response [27]. This is the first report demonstrating that co-injection of two genes expressing different antigens induces a protective response, whereas the separate injection of cp genes is not protective. In addition, immunization of BALB/c mice with combination of DNA and protein immunization can be as effective as DNA immunization which present an alternative way of immunization and vaccine development in the prevention of leishmaniasis.
Table 1 IFN-g production following cps DNA vaccination as 1st immunization and recombinant CPs (rCPa and rCPb) as booster injection in BALB/c mice Immunization time
4 weeks after first immunization 4 weeks after second immunization 8 weeks after challenge
IFN-g (pg/ml) production in response to rCPa+rCPb
Leishmania lysate
1300 9 285
800 9176
4800 9 405
1400 9224
3100 9320
2000 9398
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[10] [11]
[12]
[13]
Fig. 4. IgG1 and IgG2a profile of CP-specific antibodies induced by immunization with cps DNA. Mice were bled at week 8 after challenge and pools of 7 – 10 mice/group were analyzed for presence of CPs-reactive IgG1 and IgG2a.
Acknowledgements We wish to thank Dr J. Mauel, Dr I. Fischberg, Dr Ruddger and Dr S. Masina for their helpful advice and revision of the paper. This investigation received financial support from UNDP/World Bank/WHO Special, Program for Research and Training in Tropical Disease (TDR), ID c970556. This work was also supported by grants nos. 31-47342.96 and 31-59450.99 from the FNRS to N.F who was the recipient of the Dr Max Cloetta fellowship (Switzerland).
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