Highly conserved intracellular proteins from Leishmania have been described as antigens in natural and experimental infected mammals. The present study aimed to evaluate the antigenicity and prophylactic properties of the Leishmania infantum Poly (A) binding proteins (LiPABPs). Three different members of the LiPABP family have been described. Recombinant tools based on these proteins were constructed: recombinant proteins and DNA vaccines. The three recombinant proteins were employed for coating ELISA plates. Sera from human and canine patients of visceral leishmaniasis and human patients of mucosal leishmaniasis recognized the three LiPABPs. In addition, the protective efficacy of a DNA vaccine based on the combination of the three Leishmania PABPs has been tested in a model of progressive murine leishmaniasis: BALB/c mice infected with Leishmania major. The induction of a Th1-like response against the LiPABP family by genetic vaccination was able to down-regulate the IL-10 predominant responses elicited by parasite LiPABPs after infection in this murine model. This modulation resulted in a partial protection against L. major infection. LiPABP vaccinated mice showed a reduction on the pathology that was accompanied by a decrease in parasite burdens, in antibody titers against Leishmania antigens and in the IL-4 and IL-10 parasite-specific mediated responses in comparison to control mice groups immunized with saline or with the non-recombinant plasmid. The results presented here demonstrate for the first time the prophylactic properties of a new family of Leishmania antigenic intracellular proteins, the LiPABPs. The redirection of the immune response elicited against the LiPABP family (from IL-10 towards IFN-γ mediated responses) by genetic vaccination was able to induce a partial protection against the development of the disease in a highly susceptible murine model of leishmaniasis.
Parasites of the genus Leishmania are protozoan organisms that can infect different mammalian species including humans and dogs. Leishmania promastigote forms present in the insect vectors (phlebotomine sand flies) are transmitted to the vertebrate hosts during the blood meal [1]. After infection, parasites differentiate in the amastigote forms that multiply inside macrophages. The infected vertebrates can present a wide spectrum of clinical manifestations, globally known as leishmaniasis. Depending on the host and the parasite species, the disease can range from subclinical forms to severe symptomatic leishmaniasis [2]. In humans, there are various parasite species that cause cutaneous leishmaniasis (CL), including Leishmania major in the Old World and Leishmania braziliensis in the New World, among other species. CL is the less severe form of the disease and, in some cases, patients heal spontaneously. A percentage of the patients infected with L. braziliensis can develop a form of the disease characterized by the presence of lesions in mucosal areas, known as mucosal leishmaniasis (ML) [3]. Visceral leishmaniasis (VL) is the most severe clinical form of the disease and it is presented in human and canine patients infected with Leishmania infantum (syn. Leishmania chagasi [4]) in the Mediterranean basin and in South America, or in human patients infected with Leishmania donovani in Asia and Africa [5]. VL patients usually present hepatosplenomegaly accompanied by other clinical symptoms like fever, polyclonal hypergammaglobulinemia and anaemia [6]. Infected dogs develop similar systemic symptoms accompanied by cutaneous lesions in the mucous membranes, skin and eyes [7]. The control of infection in mammalian hosts naturally or experimentally infected with different Leishmania species depends mainly on the induction of cellular responses able to activate macrophages to eliminate the intracellular pathogens [8]. The induction of CD4+ Th1 cells specific for parasite antigens is critical to control infection due to their ability to secrete IFN-γ, but the activation of other cell subsets to produce this cytokine, like Leishmania antigen specific CD8+ T cells, is also required for a protective host response to L. major infection in the C57BL/6 mice strain. On the contrary, the generation of an IL-4 driven Th2 response after L. major infection in the susceptible BALB/c mice strain has been related to disease (reviewed in [9]). In this model of murine leishmaniasis, T cell or B cell derived IL-10 production has also been implicated in the progression of infection [10,11]. The fact that patients recovered from the disease are resistant to Leishmania reinfection can be taken as an indication that a vaccine is feasible. Regarding prophylaxis in dogs, there are three commercial vaccines against canine leishmaniasis: Leishmune, based on the Parasite Fucose-Manose-Ligand [12], Leish-Tec, a vaccine constructed by the recombinant version of the amastigote specific A2 protein [13] and CaniLeish, composed of promastigote secreted-excreted factors [14], but none of them are able to completely control the disease [15,16]. In addition, there is no vaccine against this parasite in humans. Some of the candidates for the development of human vaccines have been tested as second generation products (based on parasite or insect vector saliva defined antigens) or third generation DNA based vaccines in murine experimental models [17]. The establishment of a protective anti-Leishmania response based on these recombinant molecules requires the induction of parasite specific long-lasting memory T cells that will expand as effector T cells for the production of IFN-γ dependent responses specific for parasite antigens shortly after infection. Some experimental vaccines based on intracellular proteins that form complexes with nucleic acids, like histones or ribosomal proteins have been related with protection against the disease in different experimental models [18,19,20,21,22,23]. Poly (A) binding proteins (PABPs) are nuclear or cytosolic factors that contribute to mRNA metabolism from nuclear polyadenylation and export to translation initiation and control of RNA turnover in the cytoplasm [24]. PABPs are able to interact with the Poly (A) tail and other regions of the mRNAs as well as with other protein factors to exert their functions [25]. Three members of the PABP family have been described in L. major. Two of them, namely LmPABP1 and LmPABP2, are the cytosolic or nuclear (respectively) orthologues of the PABPs from other kinetoplastids, whereas the third one (LmPABP3) is a nuclear protein only present in the Leishmania genus among kinetoplastids [26]. Since the L. infantum PABP2 was recognized by the sera from VL symptomatic dogs [27] this work has extended the analysis of the antigenicity of all the members of the LiPABP family. The results of this work have demonstrated the antigenicity of the three LiPABPs in human and canine leishmaniasis. In addition, their immunogenicity and protective capacity when administered as a third generation DNA combined vaccine have been evaluated in a susceptible experimental model of murine leishmaniasis: BALB/c mice infected with L. major.
Female BALB/c mice (6–8 weeks old) were purchased from Harlan (Barcelona, Spain). All procedures were performed according to the Directive 2010/63/UE from the European Union and RD53/2103 from the Spanish Government and approved by the Animal Care and Use Committee at the Centro de Biología Molecular Severo Ochoa (reference CEEA-CBMSO 21/138) and the Bioethical Committee of the Consejo Superior de Investigaciones Científicas (reference 100/2014). The project was finally authorized by the Government of the Autonomous Community of Madrid (reference PROEX121/14). Promastigotes of L. infantum (MCAN/ES/1996/BCN/150/MON-1) and from L. major clone V1 (MHOM/IL/80/Friedlin) were cultured at 26°C in Schneider medium (Gibco, NY, U. S. A.) supplemented with 10% Fetal Calf Serum (FCS) (Sigma, MO, U. S. A.), 100 U/ml of penicillin and 100 μg/ml of streptomycin. Parasites were kept in a virulent state by passage in BALB/c mice. L. major amastigotes were obtained from infected popliteal lymph nodes. After transformation to the promastigote form, parasites were grown until stationary phase and then harvested for inoculation in the left hind footpad of mice or for Soluble Leishmania Antigen (SLA) preparation as described in [22]. The L. infantum PABP1 (LinJ35. V3. 5360), PABP2 (LinJ. 35. 4200) and PABP3 (LinJ. 25. 0080) (namely LiPABP1, LiPABP2 and LiPABP3, respectively) coding regions were obtained from the L. infantum genome database (www. genedb. org) using the L. major orthologous protein sequences as probes (LmjF. 35. 5040, LmjF. 35. 4130 and LmjF. 35. 4130) [26]. Coding regions were PCR amplified using genomic DNA from L. infantum (taking advantage of the lack of intron sequences in this genus) and the next specific primers: LiPABP1 forward, 5’-CGGGATCCATGGACAAGCCGATGCAGATC-3’ and reverse, 5’-CGGATATCACGCCGTCTGATGCGCCTT-3’; LiPABP2 forward, 5′- CGGGATCCATGGCCTTTACTGGTCCGAATC-3′ and reverse, 5’-CGGGATCCTCAAACGCTCATGTCTGCCAAG-3’; LiPABP3 (forward, 5’-CGGGATCCATGGTGGTCCCAGTGCAACG-3; reverse, 5’-CGGATATCAGTTGCCAGTGTGCTGCTGAAG-3’. The restriction sites included for cloning purposes (BamHI for LiPABP2 and BamHI/EcoRV for LiPABP1 and LiPABP3) are underlined. The resultant PCR products were inserted into pBluescript II SK (Stratagene. CA, U. S. A). For that purpose the PCR inserts were purified using the High Pure PCR Product Purification Kit (Roche Diagnostics GmbH, Mannheim, Germany) and the pBluescript II SK and the PCR inserts were digested with the indicated restriction enzymes (New England Biolabs, MA, U. S. A.) and ligated with the T4 DNA ligase (New England Biolabs). Positive clones were selected by a restriction analysis (using the enzymes employed for cloning) and double stranded sequenced. For subcloning purposes, the three inserts containing the coding regions were obtained by digestion with BamHI for LiPABP2 and BamHI/EcoRV for LiPABP1 and LiPABP3, and inserted in two different plasmids. For preparing genetic vaccines, the inserts were subcloned in the corresponding sites of the pcDNA3 mammalian expression vector (Invitrogen, CA, U. S. A.). The correct orientation of the LiPABP2 insert was monitored by a restriction analysis made with ApaI, an enzyme that cut the LiPABP2 coding region (positions 1651–1656 of a total length of 1756 nucleotides; Gene DB sequence LinJ. 35. 4200) and it is included in the multiple cloning site of the pcDNA3 plasmids downstream the BamHI cut site. For vaccination assays, the pcDNA3 vector and the three recombinant pcDNA3 plasmids were purified by the endotoxin-free Giga-preparation Kit (Qiagen, Hilden, Germany). Plasmids used in vaccination assays were suspended in sterile phosphate-buffered saline (PBS). For recombinant protein preparations, DNA inserts containing the different coding regions were subcloned into the BamHI (LiPABP2) or the BamHI/SmaI sites (LiPABP1 and LiPABP3) of the pQE30 prokaryotic expression vector (Qiagen). The correct LiPABP2 insert orientation was examined by restriction with SmaI enzyme, since the LiPABP2 coding region has a SmaI cut site located at nucleotides 1173–1178 of a total length of 1756 (Gene DB sequence LinJ. 35. 4200) and it is also located downstream the BamHI cut sequence in the pQE multiple cloning site. Escherichia coli (M15 strain) transfected with the recombinant plasmids were employed for over-expression of the his-tagged proteins. For that purpose, E. coli cultures were grown at 37°C in an orbital shaker to the mid-growth phase (determined by 0. 6 value of Optical Density (O. D.) at 600 nm) and the expression was induced by addition of 1 mM IPTG for 4 h at the same conditions. Since the three LiPABPs formed inclusion bodies, proteins were solubilized under denaturing conditions. For ELISA assays, proteins were purified under denaturant conditions onto Ni-nitrilotriacetic-acid-agarose (Ni-NTA, Qiagen) columns following the manufacturer’s instructions (The QIAespressionistTM; https: //www. qiagen. com/es/resources/resourcedetail? id=79ca2f7d-42fe-4d62-8676-4cfa948c9435&lang=en). Briefly, proteins were solubilized in binding buffer (20 mM Tris ClH pH 8. 0,0. 5 M NaCl, 8 M Urea, 1 mM β-mercaptoethanol) supplemented with 5 mM Imidazole, and mounted into a Ni-NTA gravity flow column. After washing in the binding buffer supplemented with 20 mM imidazole, proteins were eluted using the binding buffer supplemented with 0. 5 M imidazole. After, purified proteins were dialyzed against ELISA denaturant coating buffer (20 mM Tris ClH pH 8. 0,0. 5 M NaCl, 3 M Urea, 1 mM β-mercaptoethanol). For immunization or cells culture experiments, proteins were purified under denaturing conditions and refolded on the affinity column as described in [28] adding 20% (p/v) glycerol in those solutions that had not the denaturant agent (urea) to maintain protein solubility [29]. After purification, proteins were dialyzed against PBS plus 20% (p/v) glycerol. Polymyxin-agarose columns (Sigma) were employed to remove residual endotoxin content (<10 pg of LPS per μg of recombinant protein, measured by the Quantitative Chromogenic Limulus Amebocyte Assay QCL-1000 (BioWhittaker, MD, U. S. A.) ). Protein concentration was estimated by Bradford method using the Bio-Rad Protein assay (Bio-Rad, CA, U. S. A.). Human serum samples were obtained from clinical and parasitologically diagnosed patients. For the present study, anonymized samples were randomly selected from a sera bank (LIP-CPqGM-FIOCRUZ) built for previous independent studies conducted in Brazil in endemic areas for ML [30] or VL [31]. Canine symptomatic VL sera from L. infantum infected dogs were collected in the Extremadura region of Spain [32]. Control sera were obtained from healthy individuals (humans or dogs). For ELISA, MaxiSorp plates (Nunc, Roskilde, Denmark) were coated with 0. 2 μg of each one of the recombinant antigens (diluted in ELISA denaturant coating buffer) for 12 h at 4°C. After four washes with PBS + 0. 5% Tween 20 (PBST) wells free binding sites were blocked with blocking solution (PBST + 5% non-fat milk solution) for 1 h at room temperature (RT) and incubated with human or canine sera for 2 h at RT (1/200 dilution in blocking solution). After four washes in PBST, wells were incubated with secondary antibodies conjugated to horseradish peroxidase at 1/2,000 dilution in blocking solution for 1 h at RT (anti-dog IgG or anti-human IgG from Nordic Immunological Laboratories, Tilburg, The Netherlands). After four washes in PBST, the reaction was developed through incubation with orto-phenylenediamine 10 min in the dark, and stopped by addition of 2 N H2SO4. Optical densities were read at 450 nm in an ELISA microplate spectrophotometer (Bio-Rad). From the experimental model, sera were obtained at the beginning of the immunization (pre-immune sera), four weeks after the last doses (just before challenge with parasites) as well as seven weeks after challenge with L. major. The procedure for the ELISAs were the same indicated above but the reciprocal end-point titer (defined as the inverse of the highest serum dilution factor giving an absorbance > 0. 15) against the recombinant proteins (0. 2 μg per well) or SLA (1 μg per well) was determined by serial dilution of the sera. Anti-IgG, anti-IgG1 or anti-IgG2a (all of them at 1/2,000) horseradish peroxidase-conjugated anti-mouse immunoglobulins were used as secondary antibodies (purchased by Nordic Immunological Laboratories). To confirm that the DNA constructs were functional, COS7 cells were independently transfected with 20 μg of pcDNA3-LiPABP1, pcDNA3-LiPABP2 or pcDNA3-LiPABP3 using the Fugene reagent (Promega, WI, U. S. A.) according to the manufacturer' s protocol. Seventy-two hours post-transfection, the cells were harvested, washed two times with ice-cold PBS, and immediately lysed by the addition of Laemmli' s buffer. The proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes (Amersham, Aylesbury, United Kingdom). The blots were probed with mouse antiserum raised against the LiPABPs (1/200) by the independent immunization of the recombinant LiPABPs (three doses, 12 μg of each one) combined with 25 μg of the next two phosphorothioate-modified CpG-ODNs: (5’-TCAACGTTGA-3’ and 5’- GCTAGACGTTAGCGT-3’). Anti-IgG (1/2,000) horseradish peroxidase-conjugated anti-mouse immunoglobulins were employed as secondary antibodies (Nordic Immunological Laboratories). As control, similar blots were probed with an anti-human PABP antibody (Cell Signaling, Merck Millipore Darmstadt, Germany) and horseradish peroxidase-conjugated anti-mouse immunoglobulins (1/2,000; Nordic Immunological Laboratories) as secondary reagent. Western blots treated with anti-LiPABPs sera were revealed with the ECL Western Blotting System (GE Healthcare Life Sciences, PA, U. S. A.) and those probed with anti-human PABP were revealed with 4-chloronaphthol (Sigma). Three mice groups (n = 16 per group) were made. The LiPABP vaccinated group was inoculated in the right hind footpad with 200 μg of a mixture of the next recombinant plasmids: pcDNA3-LiPABP1, pcDNA3-LiPABP2 or pcDNA3-LiPABP3 (66. 6 μg of each plasmid). As control groups, mice were inoculated with PBS or with 200 μg of the pcDNA3 plasmid. Mice were inoculated three times (two weeks apart). Four weeks after last dose eight mice per group were euthanized, in order to obtain their spleens to be used in the analysis of the cellular immune responses induced by immunizations. Parasite challenge was carried out at the same time using the remaining mice by subcutaneous inoculation with 5 × 104 stationary-phase promastigotes of L. major into their left footpads. For the analysis of the clinical signs of infection, footpad swelling was measured with a metric caliper (expressed as thickness of the infected left footpad minus thickness of the right footpad). For parasite load determination, the spleen and the single popliteal lymph node draining the site of infection (left leg) were collected and independently processed. The complete spleens or lymph nodes were mechanically homogenized in Schneider' s medium supplemented with 20% heat-inactivated FCS, 200 U/ml penicillin and 100 μg/ml streptomycin and filtered using a cell strainer (70-μm pore size). Each homogenized sample tissue was serially diluted (1/3) in a 96-well flat-bottomed microtiter plate containing the same medium (in triplicates). The number of viable parasites was determined from the highest dilution at which promastigotes could be grown up to 10 days of incubation at 26°C as previously described [33]. Spleen cells obtained from mice inoculated with saline, with the pcDNA3 vector or with the LiPABPs combined genetic vaccine were seeded and independently cultured in RPMI complete medium at 5 × 106 cells per ml (RPMI medium (Sigma) ) supplemented with 10% heat-inactivated FCS, 20 mM L-glutamine, 200 U/ml penicillin, 100 μg/ml streptomycin and 50 μg/ml gentamicin, during 72 h at 37°C in 5% CO2. Mice (n = 8 per group) were euthanized four weeks after the last dose or seven weeks after parasite challenge. Cells were cultured alone or with some of the next stimuli: SLA, the recombinant LiPABP1, LiPABP2, LiPABP3 proteins assayed separately (all of them at 12 μg/ml of final concentration), or with a mixture of the three recombinant LiPABPs at 4 μg/ml each one. The levels of IFN-γ, IL-10 or IL-4 were determined in the culture supernatants by sandwich ELISA using monoclonal antibodies specific for mouse cytokines (capture and detection) provided in commercial kits (Pharmingen, San Diego, CA, USA), following the manufacturer’s instructions. Statistical analysis was performed using the Graph-Pad Prism program. Data were first analyzed by the D' Agostino & Pearson normality test. Parametric data were analyzed by a two-tailed Student' s t-test and non-parametric data were analyzed by a Mann Whitney test. For both test differences were considered significant when * (+) P < 0. 05, ** (++) P < 0. 01 or *** (+++) P < 0. 001.
First, the antigenicity of the members of the Leishmania PABP was determined. For this purpose, the three LiPABPs were expressed as recombinant proteins in E. coli, purified by affinity chromatography and employed for ELISA to investigate the presence of circulating antibodies against them in the sera from human and canine patients affected by VL. The three LiPABPs were recognized by Brazilian human patients suffering VL due to the infection of L. chagasi (syn. L. infantum) (Fig 1A), and by sera samples from Spanish VL symptomatic dogs infected by L. infantum (Fig 1B). On the basis of the high degree of identity observed between L. infantum and L. braziliensis counterparts (S1 Fig) the cross reactivity between LiPABPs and sera from patients affected by ML due to L. braziliensis was assayed. The reactivity of the sera from ML patients was significantly higher than that from healthy donors for the three LiPABPs (Fig 1A). From these data, it was concluded that the three members of the LiPABP family interact with the host immune system after infection in different vertebrate hosts. To evaluate the immunogenicity of the Leishmania PABPs administered as a combined DNA vaccine, the coding regions for the LiPABPs (LiPABP1, LiPABP2 and LiPABP3) were individually cloned into the eukaryotic expression vector pcDNA3. The correct expression of the DNA vaccines was tested in culture analyzing the expression of the LiPABPs in COS7 cells transfected with the recombinant plasmids (Fig 2, upper panels). Using sera from mice immunized with the recombinant versions of the parasite LiPABPs, specific protein bands were observed in the three immunoblot panels in the lanes corresponding to cell cultures transfected with the recombinant plasmids (Fig 2, upper panels, lanes 3). No reactivity was observed against proteins from COS7 cell samples un-transfected (Fig 2, upper panels, lanes 1) or transfected with the non-recombinant plasmid (Fig 2, upper panels, lanes 2). The presence of the mammalian PABP was revealed by probing similar blots with an anti-human PABP antibody (Fig 2, lower panels). The molecular weights of the parasite LiPABPs expressed by mammalian cells were close to the expected (hypothetically calculated from their amino acid sequences) and slightly lower to that observed for the recombinant LiPABPs expressed in bacteria and possessing a histidine tag in the amino-terminal region (Fig 2, upper panels, lanes 4). The lower bands detected by anti-LiPABPs antibodies may result from cleavage of the proteins expressed in the mammalian cells (Fig 2, upper panels, lanes 3). Similarly, lower bands observed in the lane 4, upper panel of the Fig 2B, may be related to partially cleaved forms of the recombinant LiPABP2 protein expressed in E. coli, since they were absent in protein samples purified under denaturant conditions and appeared when the denaturant agent was removed by dialysis against PBS (S2 Fig). Once determined the correct expression of the three Leishmania PABPs, plasmid DNAs were administered to mice as a mixed formulation (combined genetic vaccine). The cellular and humoral responses elicited by the LiPABP combined genetic vaccine were compared to those observed in two control mice groups: one receiving the vaccine saline diluent (PBS) and one receiving the pcDNA3 plasmid (as an adjuvant control). Mice vaccinated with the LiPABP genetic combined vaccine showed a positive IgG response against parasite proteins that was absent in both control groups. An IgG2a versus IgG1 dominant antibody response was observed in vaccinated mice against the antigens composing the vaccine when they were assayed by ELISA, together or separately (Fig 3A). The LiPABP1 protein was the most immunogenic of the three tested LiPABPs in BALB/c mice. No reactivity against SLA was observed in the sera of control or vaccinated mice. The systemic cellular response elicited by the immunizations was studied by the in vitro stimulation of spleen cells cultures established from control and LiPABP vaccinated BALB/c mice. A parasite LiPABP-specific secretion of IFN-γ and IL-10 was observed in mice vaccinated with the LiPABP based genetic vaccine. IFN-γ was the most abundant cytokine detected in cultures supernatants after stimulation of cells with each one of the LiPABPs or with a mixture of the three proteins (Fig 3B). Low levels of IL-4 were detected in all cell cultures supernatants assayed. Since the assays were performed in the absence of an IL-4 receptor blocking antibody it cannot be discarded the consumption of the cytokine in supernatants of cells stimulated with the antigens [34]. However, the low titer of LiPABPs specific IgG1 antibodies found in the LiPABP vaccinated mice (an IL-4-dependent isotype [35]) is reflecting the generation of poor Th2 responses. From both humoral and cellular data, it was concluded that the mixed genetic vaccine induced a predominant Th1-biased immune response specific for the three members of the LiPABP family. Since the induction of a Th1 response against different parasite antigens has been related with protection in experimental models of CL we next analyzed whether the immunization with the combined vaccine was able to induce protection against L. major infection in the highly susceptible BALB/c strain. For that purpose, mice vaccinated with the LiPABP based genetic vaccine as well as mice from control groups (inoculated with the non-recombinant plasmid or the saline diluent of the vaccine) were infected with L. major, one month after the last immunization. Mice vaccinated with the LiPABP combined vaccine showed lower footpad swelling than control groups inoculated with the vaccine diluent or the non-recombinant plasmid, from week 3 after challenge to the end of the assay (Fig 4A). The time for euthanization, seven weeks post-infection, was determined by the appearance of necrotic lesions in control groups that were absent in the LiPABP vaccinated group. In addition, LiPABP vaccinated mice showed a 1. 25-log and 1. 21-log reduction in the parasite burdens of the lymph node draining the infected footpad (popliteal lymph node) relative to saline and pcDNA3 immunized control groups, respectively (Fig 4B). Similarly, it was determined the existence of a 1. 5-log and 1. 4-log reduction in the number of viable parasite detected in the spleen of LiPABP vaccinated mice relative to both control groups (Fig 4B). Since the level of the humoral responses elicited against leishmanial antigens in mice infected with L. major has been correlated to the severity of the disease [36] the humoral response against SLA was analyzed in the three mice groups assayed. At week seven after infection the titer of SLA-specific IgG antibodies in the LiPABP vaccinated mice group was lower than the titer observed in the non-protected control groups (Fig 5A). In addition, the polarization towards a Th2 response demonstrated by titers of SLA-specific IgG1 antibodies observed in both control groups is absent in the LiPABP vaccinated group (Fig 5B). The cellular response against SLA was then analyzed using spleen cells cultures established from mice from the three tested groups. A significant decrease in the production of SLA-induced IL-10 and IL-4 was observed in the LiPABP vaccinated and protected mice, relative to both control groups (Fig 5C). Even though similar levels of IFN-γ were found in the supernatants of cultures after stimulation with SLA the ratio of IFN-γ/IL-10 and IFN-γ/IL-4 was significantly higher in the protected mice group than in animals from both control groups (Fig 5D). As control, spleen primary cultures were grown in the same conditions but in the absence of any other stimuli. The cytokine levels found in these supernatants were very low (Fig 5C; Medium). Therefore, the parasites colonizing the spleen of infected mice were not contributing to cytokine release in the in vitro stimulation assay. It was concluded that immunization with the LiPABPs combined vaccine induced a delay in the progression of CL due to L. major infection. This partial protection was correlated to the decrease of parasite specific IL-4 and IL-10 mediated responses, in the absence of changes in the magnitude of the parasite dependent IFN-γ production. The immune response against the LiPABPs after challenge was studied in mice vaccinated with the LiPABP genetic vaccine as well as in mice without a previous contact with these antigens, namely saline and pcDNA3 to analyze how infection affects the immune response elicited against the proteins composing the LiPABP vaccine in immunized mice, but also in animals without a previous contact with these proteins (saline and pcDNA3 groups). Regarding the humoral response, high IgG1 and IgG2a antibody titers were observed in the sera from LiPABP vaccinated animals after infection (Fig 6A). The predominant IgG2a response against the LiPABP family induced by vaccination was maintained after parasite challenge in this mice group (Fig 6A). The LiPABP1 (Fig 6B) was the most antigenic member of the family followed by LiPABP2 (Fig 6C), being LiPABP3 the lesser antigenic member of the family (Fig 6D). On the contrary, control groups showed a limited humoral response against the LiPABPs antigens, being the IgG1 titers higher than the IgG2a ones (Fig 6A–6D). Significant differences between both subclasses were only observed in both control groups when a mix of the three proteins were employed for coating ELISA plates (Fig 6A) or in the saline group for Leishmania LiPABP1 (Fig 6B). The cellular response induced against the LiPABPs by infection was also analyzed in the three mice groups after challenge. An IFN-γ mediated response was only observed in the animals immunized with the LiPABP genetic combined vaccine (Fig 7A). Similar levels of IL-4 and IL-10 specific for LiPABPs were observed in all mice groups (Fig 7A). Very low levels of IL-4 were observed in supernatants of cultures stimulated with the recombinant LiPABPs; less than 20 pg/ml for all groups and antigens tested. Consumption of the cytokine in supernatants of cells stimulated with the antigens can-not be discarded, since the assays were performed in the absence of an IL-4 receptor blocking antibody. A robust IL-10 mediated response was observed against the LiPABPs proteins. At week seven after infection, the three proteins were able to induce the secretion of IL-10 in the spleen cells cultures established from mice belonging to both control groups (Fig 7A). The higher values of the IL-10/ IFN-γ ratio obtained in control groups (Fig 7B) suggest that the members of the LiPABP family may be acting as an anti-inflammatory stimulus during infection. The lower value of the IL-10/ IFN-γ ratio observed in the LiPABP vaccinated and protected mice after infection indicates that the Th1 response induced by vaccination with the genetic combined vaccine against the three member of the LiPABP family was maintained after infection (Fig 7B). Very low levels of the different cytokines were found in cultures grown without stimulation (Fig 7A; Medium).
Leishmania infantum PABPs: LiPABP1 LinJ. 35. 5360 GeneDB. LiPABP2 LinJ. 35. 4200 GeneDB. LiPABP3 LinJ. 25. 0080 Gene DB. Homo sapiens PABP: GenBank: EAX07265. 1
Leishmaniases are a wide spectrum of parasite diseases caused by the infection of different species of Leishmania genus in several mammalian hosts, such as humans and dogs, among others. The induction of antibody responses against some parasite intracellular proteins implicated in different structural and functional cellular roles has been associated with parasite survival, infection progression, down-regulation of the cellular immune responses against the parasites and the development of pathology. The data presented in this work show the antigenic nature of the three members of the Leishmania infantum Poly (A) Binding Protein (LiPABP) family in different forms of natural and experimental leishmaniasis. Using a susceptible model of progressive leishmaniasis (BALB/c mice infected with Leishmania major), it was shown that the administration of a LiPABPs based combined genetic vaccine was able to induce a partial protection against the disease. Redirection of the IL-10 mediated responses elicited by the parasite LiPABPs after L. major infection towards an IFN-γ specific production by vaccination was related to protection. The inclusion of the LiPABPs in a protective vaccine formulation against leishmaniasis is discussed.
Coadministration of the Three Antigenic Leishmania infantum Poly (A) Binding Proteins as a DNA Vaccine Induces Protection against Leishmania major Infection in BALB/c Mice