- Open Access
Mitigating an undesirable immune response of inherent susceptibility to cutaneous leishmaniosis in a mouse model: the role of the pathoantigenic HISA70 DNA vaccine
Veterinary Research volume 43, Article number: 59 (2012)
Leishmania major is the major cause of cutaneous leishmaniosis (CL) outside of the Americas. In the present study we have cloned six Leishmania genes (H2A, H2B, H3, H4, A2 and HSP70) into the eukaryotic expression vector pCMVβ-m2a, resulting in pCMV-HISA70m2A, which encodes all six pathoantigenic proteins as a single polyprotein. This expression plasmid has been evaluated as a novel vaccine candidate in the BALB/c mouse model of CL. The DNA vaccine shifted the immune response normally induced by L. major infection away from a Th2-specific pathway to one of basal susceptibility. Immunization with pCMV-HISA70m2A dramatically reduced footpad lesions and lymph node parasite burdens relative to infected control mice. Complete absence of visceral parasite burden was observed in all 12 immunized animals but not in any of the 24 control mice. Moreover, vaccinated mice produced large amounts of IFN-γ, IL-17 and NO at 7 weeks post-infection (pi), and they showed lower arginase activity at the site of infection, lower IL-4 production and a weaker humoral immune response than infected control mice. Taken together, these results demonstrate the ability of the HISA70 vaccine to shift the murine immune response to L. major infection away from an undesirable, Th2-specific pathway to a less susceptible-like pathway involving Th1 and Th17 cytokine profiles.
The leishmaniases are a group of diseases caused by infection by several species of the intracellular protozoan parasites of the genus Leishmania, which are transmitted by the bite of an infected female phlebotomine sandfly. Clinical manifestations are divided into three broad categories, ranging from localized skin ulcers at the site of the sandfly bite (CL), to disfigurements (mucocutaneous leishmaniosis, MCL) and a potentially fatal disease of the viscera (visceral leishmaniosis, VL). These vector-borne diseases are a global public health problem, affecting an estimated 12 million people around the world. In fact, 1.5 million new cases of CL and 0.5 million new cases of VL are reported in humans each year.
Despite their great importance, the leishmaniases are among the most neglected tropical diseases (NTD) in the developing world. This is mainly due to their strong association with poverty: leishmaniases have not become a very profitable market for the pharmaceutical sector. However, in recent years, these NTD have come to affect not only poor countries but also developed countries as a result of poor sanitary conditions as well as migration and travel from Leishmania- endemic areas[3–5]. In addition, climate change and other environmental changes have the potential to expand the geographical distribution of phlebotomine sandfly species. Current control programs have focused their attention on vector and reservoir host control measures or mass drug distribution. Unfortunately, despite considerable progress, no vaccines are available to control any form of human leishmaniosis[7, 8].
CL is caused by Leishmania tropica and L. major in Old World countries, and by L. mexicana and L. amazonensis in Central and South America. In this disease, infection of mammalian host phagocytes results in either subclinical infection or subacute to chronic disease characterized by lesions and scarring on exposed areas of the skin. Consequently, although the cutaneous form is not a lethal disease, people living with CL face significant social stigma. In addition, failure to treat patients with CL can give Leishmania parasites time to leave the skin lesions and invade the bloodstream to spread systemically in the host. Moreover, depending on the species of Leishmania involved, cutaneous disease can progress to destructive MCL. Therefore, local or systemic treatment is important for shortening the disease duration, managing lesions, especially in the face, improving the cosmetic aspects of scarring, and avoiding the enormous social stigma[10, 11]. Treatments with antimonial compounds and amphotericin B show variable efficacy, and they are also toxic and expensive. Clearly, alternative treatment strategies and new prophylactic vaccines are needed.
Genetic vaccination is one option to efficiently prime specific, Th1 mediated host resistance against intracellular pathogens. The efficacy of DNA vaccines against experimental leishmaniosis has recently been reviewed. Leishmania is an intracellular parasite of mammalian phagocytic cells, such as macrophages and dendritic cells (DC). The outcome of the infection depends on the type of host immune response elicited. These phagocytic cells can control Leishmania infection when a Type 1 T helper (Th1) response is mounted, leading to the induction of inducible nitric oxide synthase (iNOS) and NO production, which is the main Leishmania killer molecule in the murine system. However, Leishmania can develop several immune evasion tricks to persist in mammalian phagocytic cells. Cytokines released by Type 2 T helper (Th2) cells increase host cell arginase activity, producing polyamines that the pathogen uses for survival. Therefore, the relative strength of the Th1 and Th2 responses remains the governing principle in L. major immunity. Insights into this theory of Th1/Th2 balance have come from studies in BALB/c mice, which show T-cell-mediated susceptibility to L. major infection. In this mouse model, development and progression of the disease requires sustained production of IL-4 by Th2 cells, whereas the Th1 response mediated by IL-12, IFN-γ, and TNF-α is associated with lesion resolution and control of parasite spread[11, 17].
Recent developments are being used to investigate parasite virulence factors, elucidate immune regulatory mechanisms and contribute to the development of novel therapeutics and vaccines for the leishmaniases[18, 19]. Recognition of Leishmania antigens by sera from patients and dogs suffering leishmaniosis is one of the methods most commonly used for diagnosis and identification of vaccine candidates against leishmaniosis[20–25]. Over the last decade, analysis of a Leishmania cDNA library and its successive fractionation into smaller libraries has resulted in the identification of novel protective antigens[26, 27]. The sequence of events during Leishmania infection and the relevance of two distinct sets of parasite molecules have recently been reviewed. Inside the susceptible mammalian host, Leishmania parasites produce surface, secreted and excreted antigens that help establish infection by preventing premature damage in both parasite and host cell. Later, intracellular parasite molecules are exposed to the host immune system. These intracellular antigens are considered “pathoantigenic” molecules, since they elicit immunological responses that contribute to disease pathology[29, 30].
These pathoantigens were identified by Western blot analysis and/or immunoscreening of Leishmania expression libraries using patient sera. They have attracted considerable attention because of two remarkable properties. First, they are recognized specifically by antibodies that do not cross-react with the host counterparts. This specificity, which makes pathoantigens a valuable tool in diagnosis, is due to the fact that their antigenic determinants lie in divergent regions of the sequence, as has been shown for Leishmania pathoantigens, namely nucleosome-forming histones (H2A, H2B, H3 and H4)[21, 31], the amastigote-specific A2 protein, and the 70-kDa heat shock protein (HSP70)[20, 24, 25, 33]. Second, immunization studies show that some of these pathoantigens can decrease the pathology-inducing Th2 immune response and increase a therapeutically more appropriate Th1-biased parasite-specific immune response[34–36].
The main goal of our project is to develop a pathoantigenic vaccine able to provide cross-protection against multiple Leishmania species. In order to design an improved vaccine against CL, we have cloned the full-length coding sequences of six Leishmania pathoantigenic genes (H2A, H2B, H3, H4, A2 and HSP70) into a mammalian expression vector that expresses the resulting polyprotein, called HISA70, in mammalian cells. The resulting plasmid, named pCMV-HISA70m2A, was used to immunize BALB/c mice against virulent L. major challenge.
Materials and methods
DNA sequences of Leishmania genes were retrieved from the GeneDB and GenBank databases as follows: H2A (Lin J21.V3.1160), H2B (Lin.J09.V3.1410), H3 (LinJ10.V3.0920), H4 (Lin J31.V3.3320), A2 (GenBank S69693) and HSP70 (GenBank CAA69282.1). Then mouse codon-optimized versions of the sequences were generated using the GeneOptimizer® expert software system (Geneart AG) as described elsewhere. The optimized sequences were synthesized chemically by GeneArt as a single coding region of 4101 bp, named HISA70m. This sequence encodes a polyprotein of 1367 amino acid residues comprising, from N- to C-terminus, H2A-H2B-H3-H4-A2-HSP70. To facilitate further plasmid construction, Xho I and Not I sites were inserted, respectively, upstream and downstream of the HISA70m fusion gene, and this expression cassette was cloned into the eukaryotic expression plasmid pCMVβ-m2A. Design of the plasmid DNA vaccine is shown in Figure1A.
The expression plasmid pCMV-HISA70m2A and empty vector pCMVβ-m2A were purified using the EndoFree plasmid Mega kit (Qiagen, Hilden, Germany) according to the manufacturer's recommended protocol. The final pellets of endotoxin-free DNA plasmid were resuspended in sterile PBS and stored at −20°C until their use in immunization.
Transfection of plasmid constructs and Western blotting
Expression of HISA70 was detected in mammalian cells by transiently transfecting pCMV-HISA70m2A into CHO-K1 cells using Lipofectamine LTX and PLUS Reagent (Invitrogen, San Diego, CA, USA) according to the manufacturer's instructions. Briefly, CHO-K1 cells were cultured at a density of 4 × 104 cells per well in 24-well plates in F-12K Nutrient Mixture with Kaighn’s modification (Invitrogen) containing 10% FCS and antibiotics; cells were transfected when they reached 50–80% confluence. Transfected cells were incubated 24 h at 37°C with 5% CO2. Then the cells were harvested and lysed by addition of 200 μL per well of Laemmli’s buffer (Bio-Rad) containing β-mercaptoethanol. Lysates (15 μL) were resolved by SDS-PAGE. Protein bands were electrophoretically transferred to a PVDF membrane (GE Healthcare, Madrid, Spain), which was blocked for 1 h in blocking buffer (10% non-fat milk in PBS-Tween-20). To detect the antigen blotted on the membrane, a primary antibody against L. infantum H2A was added at an appropriate dilution (1:200) and incubated with the membrane. This antibody was purified from a pool of anti-histone-positive sera from dogs suffering from VL. Subsequently the membrane was blotted with a horseradish peroxidase-conjugated rabbit anti-dog IgG secondary antibody (1:2000; Cultek, Nordic Immunological Laboratories, Madrid, Spain).
Experimental animals, parasites and preparation of soluble antigen
Eight-week-old female BALB/c mice were purchased from Harlan Interfauna Ibérica (Barcelona, Spain). The animals were maintained under standard conditions approved by the Ethics Committee of Complutense University of Madrid. L. major (clone V1: MHOM/IL/80/Friedlin) was cultured at 26°C in Schneider’s medium (Sigma-Aldrich, Madrid, Spain). Soluble Leishmania antigen (SLA) was prepared from stationary cultures of L. major promastigotes as previously described.
Vaccination and infection of mice
Two groups of mice (n = 6) were immunized subcutaneously (s.c.) in the right footpad with 100 μg (in a volume of 40 μL) of the mammalian expression vector pCMVβ-m2A alone or with pCMV-HISA70m2A on days −60, -45 and −30. In parallel, a group of control mice (n = 6) was inoculated with PBS alone using the same procedure. On day 0 all groups of mice were infected by s.c. injection in the left footpad with 5 × 103 metacyclic L. major promastigotes in a volume of 30 μL. Metacyclic forms were previously isolated from stationary cultures by negative selection using peanut agglutinin (Vector Laboratories, Barcelona, Spain). The course of infection was monitored weekly by measuring footpad swelling with a caliper. Clinical signs of ulceration were also assessed. Mice were euthanized by cervical dislocation at 7 weeks post-infection (pi), because at this time the lesions from non-vaccinated groups were larger than 4 mm in diameter or showed signs of ulceration. Draining lymph nodes (DLN) and spleens were removed from the euthanized mice. The experiments were carried out twice in order to evaluate the reproducibility of the approach.
Generation of bone marrow-derived murine DC
Bone marrow cells were obtained from the femurs and tibiae of naïve BALB/c mice and cultured in the presence of 20 ng/mL murine granulocyte macrophage colony-stimulating factor (GM-CSF; PeproTech, London, UK), as previously described. On day 7, bone marrow-derived DC were plated at 1 × 106 cells/mL in 6-well plates and primed in the presence or absence of SLA (50 μg/mL). DC were collected at 24 h after pulsing with SLA and used for in vitro stimulation of T cells as described below.
In vitro cell culture for determination of cytokine response and nitric oxide/nitrite production
At sacrifice 7 weeks pi, popliteal-DLN were harvested and prepared as single-cell suspensions as follows. DLN cells were washed, resuspended at a final concentration of 2 × 106/mL in complete medium (DMEM supplemented with 10% heat-inactivated FCS, 2 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin), and plated at 1 mL/well in 24-well plates containing bone marrow-derived DC (5 T cells : 1 DC) that had been left unstimulated or pulsed with SLA as described above. Cells were co-cultured for 96 h at 37°C and 5% CO2, and the culture supernatant was collected and stored at −20°C. Production of antigen-specific IFN-γ (Diaclone, Besançon, France), IL-4 (eBioscience, Barcelona, Spain) and IL-17 (R&D Systems, Madrid, Spain) was determined by ELISA according to the manufacturers’ suggested protocols. Levels of nitrite, which is a byproduct of nitric oxide production, were measured in culture supernatants using the Griess assay as previously described. Briefly, 100 μL of culture supernatants were mixed with an equal volume of Griess reagent (Sigma-Aldrich) and incubated at room temperature for 10 min. Absorbance was then measured at 540 nm.
Arginase activity assay
Arginase activity at the site of lesions was determined ex vivo using 5–10 mL of footpad homogenate as previously described[45, 46]. Briefly, L. major-infected footpads were rinsed in ethanol and homogenized by hand in PBS containing 100 U/mL penicillin and 100 μg/mL streptomycin. The homogenate was first centrifuged at low speed (50 g) for 5 min to remove large tissue debris, and the supernatant was then centrifuged again in PBS at high speed (1811 g) for 15 min to pellet phagocytic cells and amastigotes. Both pellets were incubated for 30 min in 2 mL of lysis buffer (0.1 M Tris–HCl, 300 μM NaCl, 1 μM PMSF, 1% Triton X-100) and the lysate was assayed for arginase activity as previously described[47, 48]. One unit of enzyme activity is defined as the amount of enzyme that catalyzes the formation of 1 mmol of urea/min.
Estimation of parasite burden
Parasite burden in the ipsilateral popliteal-DLN and spleen was determined at 7 weeks pi by limiting dilution culture. Briefly, the organs were harvested and a suspension was prepared by grinding the tissue in 1 mL of Schneider’s medium (Sigma-Aldrich) containing 20% FCS in the presence of streptomycin and penicillin. Four-fold serial dilutions of the homogenized tissue suspensions were plated in a 96-well culture plate and incubated at 26°C for 10 days. Wells were examined for viable promastigotes. The reciprocal of the highest dilution factor that was positive for promastigotes was considered to be the number of Leishmania parasites per mg of tissue.
Humoral immune response
Leishmania-specific antibodies were quantified by ELISA. Standard plates were coated overnight at 4°C with 100 μL of SLA (2 μg/mL) diluted in PBS. Afterwards, wells were washed with PBS supplemented with 0.05% (v/v) Tween-20 and blocked with 2% (w/v) BSA in PBS. Sera were serially diluted in order to determine the titer, which was defined as the inverse of the highest serum dilution factor giving an absorbance > 0.2. Secondary antibodies were peroxidase-conjugated goat anti-mouse IgG, IgG1 and IgG2a (1:500, SouthernBiotech, Madrid, Spain). After washing and addition of peroxidase substrate (ABTS, Roche Diagnostics, Madrid, Spain), sample absorbance was measured at 405 nm.
Since statistical analysis showed that measurements followed the standard normal distribution, Student’s t-test was used to evaluate the significance of differences between means in the control and experimental groups; means were the average from two independent experiments. Differences were considered significant when P < 0.05. Statistical analyses were performed using SigmaPlot software (version 12.2, Systat Software).
HISA70 protein can be expressed in CHO-K1 cells
Expression of recombinant proteins by host mammalian cells transfected with plasmid DNA is an essential requirement for stimulating the host immune system. Expression of HISA70 in CHO-K1 cells transfected with pCMV-HISA70m2A was easily detected using an antibody against L. infantum H2A (Figure1B).
Vaccination with pCMV-HISA70m2A confers protection against L. major infection in BALB/c mice
As expected, immunization of mice with pCMVβ-m2A alone had no effect on lesion development during the course of infection. These mice succumbed to progressive disease. Indeed, the size of footpad swellings (Figure2A) and parasite burdens (Figure2B) in the popliteal-DLN and spleen of these mice were not significantly different from those of control mice (PBS) at 7 weeks pi. In contrast, mice immunized with pCMV-HISA70m2A did not develop lesions at the site of infection or, in some cases, they developed significantly smaller footpad lesions up to 5 weeks after challenge with L. major, with no signs of ulcer formation or necrotic tissue. Moreover, the numbers of parasites in popliteal-DLN were significantly lower in mice immunized with pCMV-HISA70m2A than in control mice (PBS or pCMVβ-m2A alone), and immunized mice did not show the visceralization observed in the control animals (Figure2B).
Vaccination with pCMV-HISA70m2A redirects the L. major-specific Th2 immune response to a less susceptible-like phenotype
In order to elucidate the type of immune responses elicited by L. major infection, we assessed the production of IFN-γ, IL-17 and IL-4 in the popliteal-DLN after stimulation with SLA. As shown in Table1, DLN cells of mice vaccinated with pCMV-HISA70m2A produced significantly lower levels of specific IL-4 than those of control mice, but they also produced significantly larger amounts of IFN-γ and IL-17 at 7 weeks pi. In contrast, DLN cells of mice immunized with pCMVβ-m2A alone produced substantial amounts of IL-4, as well as levels of IFN-γ and IL-17 similar to those of non-vaccinated mice.
Competition between iNOS and arginase enzymes in phagocytic cells contribute to the outcome of L. major infection
Our data showed that arginase activity at the site of infection was significantly lower in vaccinated mice than in control animals (Figure3A). We also measured iNOS activity indirectly by assaying nitrite production after stimulating DLN cells with SLA-pulsed DC. We found significantly higher nitrite levels in cells of mice vaccinated with pCMV-HISA70m2A than in those of non-vaccinated mice (Figure3B). Conversely, no significant difference between control and pCMVβ-m2A immunized mice was found at 7 weeks pi.
Characterization of the specific humoral response in immunized mice
Previous studies in the CL mouse model have reported that the relative production of immunoglobulin isotypes correlates with the relative strength of induction of Th1- and Th2-biased immune responses[50, 51]. To characterize the humoral immune response to L. major infection, we measured levels of IgG1 and IgG2a isotypes in sera of control and immunized mice at 7 weeks pi (Figure4A). This analysis revealed that levels of Leishmania-specific IgG1 antibodies were significantly lower in mice immunized with pCMV-HISA70m2A than in control mice. Consequently, the mean Th1/Th2 humoral ratio after infection was significantly higher in mice vaccinated with pCMV-HISA70m2A than in the two control groups (Figure4B).
Attempts to develop immunization protocols to protect against Leishmania infection have met with mixed success. A series of studies have documented resistance to infection after immunization with Leishmania pathoantigens. Table2 summarizes data from preclinical studies of potential vaccines in animal models; these vaccines involve one or more of the six intracellular pathoantigens used to design the HISA70 vaccine in the present work (H2A, H2B, H3, H4, A2 and HSP70).
Immunization studies in mice using various combinations of Leishmania nucleosomal histones, either encoded in DNA plasmids or pulsed into dendritic cells, have demonstrated different levels of protection against CL[40, 53] and VL. Unfortunately, only partial reduction of parasite burden in lymph nodes has been reported against canine VL. Recent studies have suggested that the presence or absence of immunodominant epitopes on histones strongly influences Leishmania parasite persistence, and that Treg cells mediate this effect, suggesting the complexity of the immunization process[52, 54].
The immunogenic properties of Leishmania A2 antigen have been extensively described[69–73]. Several vaccine approaches using the A2 antigen have conferred different levels of protection against murine VL[58–60, 62–64]. Vaccination of dogs using A2 confers partial protection to canine VL, which is significant because dogs are the main domestic reservoir of viscerotropic species of Leishmania. Furthermore, immunizing mice with a combination of A2 protein and IL-12 or with an expression vector encoding A2 protected them against challenge with L. amazonensis, the causative agent of CL in Latin America[57, 58]. Curiously, no studies have analyzed the ability of A2 antigen vaccines to protect against L. major infection, which is responsible for CL in humans in the Old World. The most likely reason is that the L. donovani A2 protein coding sequence, which is the only one tested so far in vaccines, is not present in the genome of all Leishmania species and appears to be absent from L. major and L. tropica.
HSP are abundant intracellular proteins present in all cells. Since their discovery, HSP have been assigned various functions, such as folding, assembly, protein transport and thermotolerance. After exposure to various forms of shock, such as exposure to high temperatures (“heat shock”), toxins, oxidative stress, and glucose deprivation, HSP levels can rise to protect cellular proteins against denaturation. HSP also bind antigenic peptides and interact with antigen-presenting cells. Microbial HSP have been implicated in the induction of the innate and adaptive arms of the immune response[76, 77]. Interestingly, when HSP bind to antigenic peptides, the resulting complexes can prime T cell immunity specifically against the bound peptides, but not against HSP themselves. Indeed, vaccines based on HSP-peptide complexes have recently been considered for immunotherapy against cancers and infectious diseases.
Among HSP, HSP70 stands out because its complexes with antigenic peptides can generate a peptide-specific cellular immune response four orders of magnitude more efficient than the peptide alone. In the same way, Leishmania GP63 antigen administered without adjuvant elicited partial protection in mice, but when it was coadministered with HSP70, it conferred protection against VL[67, 68]. Furthermore, a cocktail of HSP70 and HSP83 seems to reduce hepatic parasite burden against murine VL. In contrast, other authors failed to demonstrate protection after immunization with HSP70 alone in a murine CL model. These findings are consistent with the role of HSP70 as an adjuvant in antigen presentation, and they suggest that HSP70 must be bound to antigen to induce an immune response. This makes sense, because HSP are self-antigens, so they should not elicit immune responses against themselves[78, 80]. Thus, vaccines based on complexes of HSP and microbial peptides show promise as safe and effective therapies against intracellular pathogens.
Given the immunogenicity of all six intracellular Leishmania pathoantigens, we developed a DNA vaccine that encodes all six proteins as a single polyprotein (HISA70). In the present study we found that DNA immunization of BALB/c mice with pCMV-HISA70m2A induced protection against L. major infection. Normally, after Leishmania invasion in BALB/c mice, infected phagocytic cells are stimulated by a Th2-type signal, cytokine IL-4, to express arginase. This inducible enzyme initiates L-arginine degradation, leading to the synthesis of polyamines, which are necessary for Leishmania parasite growth in infected cells. In the present study, popliteal-DLN of vaccinated mice showed low levels of Leishmania-specific IL-4 at 7 weeks pi, which led to significantly lower arginase activity at the site of the lesion in mice vaccinated with pCMV-HISA70m2A than in control mice. This in turn allowed the development of a protective anti-Leishmania Th1 response mediated by NO-dependent IFN-γ production. This was in accordance with previous findings that phagocytic cells can control Leishmania infection when a Th1 response is mounted and high levels of NO are produced. NO is the most important Leishmania killer molecule in the mouse, and levels of nitrite, a byproduct of NO synthesis, were significantly higher in the vaccinated mouse than in the control animals.
In addition to nitrite, our mice vaccinated with pCMV-HISA70m2A expressed significantly higher levels of specific IL-17 than did the control mice. Our finding of elevated IL-17 levels in vaccinated mice is consistent with previous studies in which parasite-specific cellular responses shifted towards Th1 and Th17 mechanisms of protective immunity in both VL and CL murine models[82, 83]. IL-17 favors the production of chemokines that mediate the recruitment of neutrophils as well as Th1 cells[84, 85]. Th17 cells secrete IL-17, IL-21, and IL-22, which exert strong proinflammatory effects that play a crucial role in fighting Gram-negative bacteria, fungi, and some protozoa. Indeed, IL-17 mediates control of parasitemia in mice infected with Trypanosoma congolense.
We found that the HISA70 vaccine did not enhance the humoral response in mice. In addition, we assessed the relative production of IgG2a and IgG1 isotypes, since these have been widely used as markers, respectively, of the induction of Th1- and Th2-type immune responses. Vaccinated mice showed a lower titer of the anti-SLA IgG1 isotype than did control mice. In contrast, SLA-specific IgG1 antibodies predominated in control mice. Thus, vaccination increased the ratio of IgG2a/IgG1 in protected mice.
In conclusion, vaccination with pCMV-HISA70m2A successfully promoted Th1- and Th17-type immune responses, thereby mitigating the Th2 response caused by L. major infection and creating a resistant-like phenotype. As a result, vaccinated mice developed much fewer and slower-growing lesions than did control mice, and showed a lower parasite burden at the site of infection, with no visceralization. These findings suggest the usefulness of HISA70 for designing vaccines with improved protective efficacy against murine CL.
Complutense University of Madrid has filed a patent on the HISA70 as a DNA vaccine against leishmaniosis.
Desjeux P: Leishmaniasis: current situation and new perspectives. Comp Immunol Microbiol Infect Dis. 2004, 27: 305-318. 10.1016/j.cimid.2004.03.004.
Bethony JM, Cole RN, Guo X, Kamhawi S, Lightowlers MW, Loukas A, Petri W, Reed S, Valenzuela JG, Hotez PJ: Vaccines to combat the neglected tropical diseases. Immunol Rev. 2011, 239: 237-270. 10.1111/j.1600-065X.2010.00976.x.
Ready PD: Leishmaniasis emergence in Europe. Euro Surveill. 2010, 15: 19505-
Shaw JJ, De Faria DL, Basano SA, Corbett CE, Rodrigues CJ, Ishikawa EA, Camargo LM: The aetiological agents of American cutaneous leishmaniasis in the municipality of Monte Negro, Rondonia state, western Amazonia, Brazil. Ann Trop Med Parasitol. 2007, 101: 681-688. 10.1179/136485907X229103.
Abadir A, Patel A, Haider S: Systemic therapy of New World cutaneous leishmaniasis: a case report and review article. Can J Infect Dis Med Microbiol. 2011, 21: e79-e83.
Ready PD: Leishmaniasis emergence and climate change. Rev Sci Tech. 2008, 27: 399-412.
Murray HW, Berman JD, Davies CR, Saravia NG: Advances in leishmaniasis. Lancet. 2005, 366: 1561-1577. 10.1016/S0140-6736(05)67629-5.
Okwor I, Uzonna J: Vaccines and vaccination strategies against human cutaneous leishmaniasis. Hum Vaccin. 2009, 5: 291-301. 10.4161/hv.5.5.7607.
Nakkash-Chmaisse H, Makki R, Nahhas G, Knio K, Nuwayri-Salti N: Detection of Leishmania parasites in the blood of patients with isolated cutaneous leishmaniasis. Int J Infect Dis. 2011, 15: e491-e494. 10.1016/j.ijid.2011.03.022.
Blum J, Desjeux P, Schwartz E, Beck B, Hatz C: Treatment of cutaneous leishmaniasis among travellers. J Antimicrob Chemother. 2004, 53: 158-166. 10.1093/jac/dkh058.
Ameen M: Cutaneous leishmaniasis: advances in disease pathogenesis, diagnostics and therapeutics. Clin Exp Dermatol. 2010, 35: 699-705. 10.1111/j.1365-2230.2010.03851.x.
Reithinger R, Dujardin JC, Louzir H, Pirmez C, Alexander B, Brooker S: Cutaneous leishmaniasis. Lancet Infect Dis. 2007, 7: 581-596. 10.1016/S1473-3099(07)70209-8.
Gurunathan S, Klinman DM, Seder RA: DNA vaccines: immunology, application, and optimization*. Annu Rev Immunol. 2000, 18: 927-974. 10.1146/annurev.immunol.18.1.927.
Dumonteil E: DNA vaccines against protozoan parasites: advances and challenges. J Biomed Biotechnol. 2007, 2007: 90520-
Wanasen N, Soong L: L-arginine metabolism and its impact on host immunity against Leishmania infection. Immunol Res. 2008, 41: 15-25. 10.1007/s12026-007-8012-y.
Das P, Lahiri A, Chakravortty D: Modulation of the arginase pathway in the context of microbial pathogenesis: a metabolic enzyme moonlighting as an immune modulator. PLoS Pathog. 2010, 6: e1000899-10.1371/journal.ppat.1000899.
Iwasaki A: The importance of CD11b + dendritic cells in CD4+ T cell activation in vivo: with help from interleukin 1. J Exp Med. 2003, 198: 185-190. 10.1084/jem.20030737.
Beattie L, Evans KJ, Kaye PM, Smith DF: Transgenic Leishmania and the immune response to infection. Parasite Immunol. 2008, 30: 255-266. 10.1111/j.1365-3024.2008.01020.x.
Evans KJ, Kedzierski L: Development of vaccines against visceral leishmaniasis. J Trop Med. 2012, 2012: 892817-
Quijada L, Requena JM, Soto M, Alonso C: During canine viscero-cutaneous leishmaniasis the anti-Hsp70 antibodies are specifically elicited by the parasite protein. Parasitology. 1996, 112: 277-284. 10.1017/S0031182000065793.
Soto M, Requena JM, Gomez LC, Navarrete I, Alonso C: Molecular characterization of a Leishmania donovani infantum antigen identified as histone H2A. Eur J Biochem. 1992, 205: 211-216. 10.1111/j.1432-1033.1992.tb16770.x.
Soto M, Requena JM, Quijada L, Perez MJ, Nieto CG, Guzman F, Patarroyo ME, Alonso C: Antigenicity of the Leishmania infantum histones H2B and H4 during canine viscerocutaneous leishmaniasis. Clin Exp Immunol. 1999, 115: 342-349. 10.1046/j.1365-2249.1999.00796.x.
Soto M, Requena JM, Garcia M, Gomez LC, Navarrete I, Alonso C: Genomic organization and expression of two independent gene arrays coding for two antigenic acidic ribosomal proteins of Leishmania. J Biol Chem. 1993, 268: 21835-21843.
MacFarlane J, Blaxter ML, Bishop RP, Miles MA, Kelly JM: Identification and characterisation of a Leishmania donovani antigen belonging to the 70-kDa heat-shock protein family. Eur J Biochem. 1990, 190: 377-384. 10.1111/j.1432-1033.1990.tb15586.x.
Skeiky YA, Benson DR, Guderian JA, Whittle JA, Bacelar O, Carvalho EM, Reed SG: Immune responses of leishmaniasis patients to heat shock proteins of Leishmania species and humans. Infect Immun. 1995, 63: 4105-4114.
Melby PC, Ogden GB, Flores HA, Zhao W, Geldmacher C, Biediger NM, Ahuja SK, Uranga J, Melendez M: Identification of vaccine candidates for experimental visceral leishmaniasis by immunization with sequential fractions of a cDNA expression library. Infect Immun. 2000, 68: 5595-5602. 10.1128/IAI.68.10.5595-5602.2000.
Martins DR, Jeronimo SM, Donelson JE, Wilson ME: Leishmania chagasi T-cell antigens identified through a double library screen. Infect Immun. 2006, 74: 6940-6948. 10.1128/IAI.02032-05.
Santarem N, Silvestre R, Tavares J, Silva M, Cabral S, Maciel J, Cordeiro-da-Silva A: Immune response regulation by leishmania secreted and nonsecreted antigens. J Biomed Biotechnol. 2007, 2007: 85154-
Chang KP, McGwire BS: Molecular determinants and regulation of Leishmania virulence. Kinetoplastid Biol Dis. 2002, 1: 1-10.1186/1475-9292-1-1.
Chang KP, Reed SG, McGwire BS, Soong L: Leishmania model for microbial virulence: the relevance of parasite multiplication and pathoantigenicity. Acta Trop. 2003, 85: 375-390. 10.1016/S0001-706X(02)00238-3.
Soto M, Requena JM, Morales G, Alonso C: The Leishmania infantum histone H3 possesses an extremely divergent N-terminal domain. Biochim Biophys Acta. 1994, 1219: 533-535. 10.1016/0167-4781(94)90082-5.
Charest H, Matlashewski G: Developmental gene expression in Leishmania donovani: differential cloning and analysis of an amastigote-stage-specific gene. Mol Cell Biol. 1994, 14: 2975-2984.
Quijada L, Requena JM, Soto M, Gomez LC, Guzman F, Patarroyo ME, Alonso C: Mapping of the linear antigenic determinants of the Leishmania infantum hsp70 recognized by leishmaniasis sera. Immunol Lett. 1996, 52: 73-79. 10.1016/0165-2478(96)02585-0.
Iborra S, Soto M, Carrion J, Alonso C, Requena JM: Vaccination with a plasmid DNA cocktail encoding the nucleosomal histones of Leishmania confers protection against murine cutaneous leishmaniosis. Vaccine. 2004, 22: 3865-3876. 10.1016/j.vaccine.2004.04.015.
de Oliveira CI, Nascimento IP, Barral A, Soto M, Barral-Netto M: Challenges and perspectives in vaccination against leishmaniasis. Parasitol Int. 2009, 58: 319-324. 10.1016/j.parint.2009.07.013.
Nagill R, Kaur S: Vaccine candidates for leishmaniasis: a review. Int Immunopharmacol. 2011, 11: 1464-1488. 10.1016/j.intimp.2011.05.008.
Raab D, Graf M, Notka F, Schodl T, Wagner R: The GeneOptimizer Algorithm: using a sliding window approach to cope with the vast sequence space in multiparameter DNA sequence optimization. Syst Synth Biol. 2010, 4: 215-225. 10.1007/s11693-010-9062-3.
Bauer H, Darji A, Chakraborty T, Weiss S: Salmonella-mediated oral DNA vaccination using stabilized eukaryotic expression plasmids. Gene Ther. 2005, 12: 364-372. 10.1038/sj.gt.3302423.
Carrion J, Folgueira C, Alonso C: Transitory or long-lasting immunity to Leishmania major infection: the result of immunogenicity and multicomponent properties of histone DNA vaccines. Vaccine. 2008, 26: 1155-1165. 10.1016/j.vaccine.2007.12.051.
Scott P, Pearce E, Natovitz P, Sher A: Vaccination against cutaneous leishmaniasis in a murine model. I. Induction of protective immunity with a soluble extract of promastigotes. J Immunol. 1987, 139: 221-227.
Sacks DL, Melby PC: Animal models for the analysis of immune responses to leishmaniasis. Curr Protoc Immunol. 2001, 19: 19.2-
Lutz MB, Kukutsch N, Ogilvie AL, Rossner S, Koch F, Romani N, Schuler G: An advanced culture method for generating large quantities of highly pure dendritic cells from mouse bone marrow. J Immunol Methods. 1999, 223: 77-92. 10.1016/S0022-1759(98)00204-X.
Ding AH, Nathan CF, Stuehr DJ: Release of reactive nitrogen intermediates and reactive oxygen intermediates from mouse peritoneal macrophages. Comparison of activating cytokines and evidence for independent production. J Immunol. 1988, 141: 2407-2412.
Kropf P, Fuentes JM, Fahnrich E, Arpa L, Herath S, Weber V, Soler G, Celada A, Modolell M, Muller I: Arginase and polyamine synthesis are key factors in the regulation of experimental leishmaniasis in vivo. FASEB J. 2005, 19: 1000-1002.
Muleme HM, Reguera RM, Berard A, Azinwi R, Jia P, Okwor IB, Beverley S, Uzonna JE: Infection with arginase-deficient Leishmania major reveals a parasite number-dependent and cytokine-independent regulation of host cellular arginase activity and disease pathogenesis. J Immunol. 2009, 183: 8068-8076. 10.4049/jimmunol.0803979.
Corraliza IM, Campo ML, Soler G, Modolell M: Determination of arginase activity in macrophages: a micromethod. J Immunol Methods. 1994, 174: 231-235. 10.1016/0022-1759(94)90027-2.
Garrido VV, Dulgerian LR, Stempin CC, Cerban FM: The increase in mannose receptor recycling favors arginase induction and Trypanosoma cruzi survival in macrophages. Int J Biol Sci. 2011, 7: 1257-1272.
Buffet PA, Sulahian A, Garin YJ, Nassar N, Derouin F: Culture microtitration: a sensitive method for quantifying Leishmania infantum in tissues of infected mice. Antimicrob Agents Chemother. 1995, 39: 2167-2168. 10.1128/AAC.39.9.2167.
Scott P: The role of TH1 and TH2 cells in experimental cutaneous leishmaniasis. Exp Parasitol. 1989, 68: 369-372. 10.1016/0014-4894(89)90120-3.
Coffman RL, Lebman DA, Rothman P: Mechanism and regulation of immunoglobulin isotype switching. Adv Immunol. 1993, 54: 229-270.
Chenik M, Louzir H, Ksontini H, Dilou A, Abdmouleh I, Dellagi K: Vaccination with the divergent portion of the protein histone H2B of Leishmania protects susceptible BALB/c mice against a virulent challenge with Leishmania major. Vaccine. 2006, 24: 2521-2529. 10.1016/j.vaccine.2005.12.027.
Carrion J, Nieto A, Soto M, Alonso C: Adoptive transfer of dendritic cells pulsed with Leishmania infantum nucleosomal histones confers protection against cutaneous leishmaniosis in BALB/c mice. Microbes Infect. 2007, 9: 735-743. 10.1016/j.micinf.2007.02.018.
Carrion J: Mechanisms of immunity to Leishmania major infection in mice: the contribution of DNA vaccines coding for two novel sets of histones (H2A-H2B or H3-H4). Comp Immunol Microbiol Infect Dis. 2011, 34: 381-386. 10.1016/j.cimid.2011.06.002.
Saldarriaga OA, Travi BL, Park W, Perez LE, Melby PC: Immunogenicity of a multicomponent DNA vaccine against visceral leishmaniasis in dogs. Vaccine. 2006, 24: 1928-1940. 10.1016/j.vaccine.2005.10.052.
Carrion J, Folgueira C, Alonso C: Immunization strategies against visceral leishmaniosis with the nucleosomal histones of Leishmania infantum encoded in DNA vaccine or pulsed in dendritic cells. Vaccine. 2008, 26: 2537-2544. 10.1016/j.vaccine.2008.03.003.
Coelho EA, Tavares CA, Carvalho FA, Chaves KF, Teixeira KN, Rodrigues RC, Charest H, Matlashewski G, Gazzinelli RT, Fernandes AP: Immune responses induced by the Leishmania (Leishmania) donovani A2 antigen, but not by the LACK antigen, are protective against experimental Leishmania (Leishmania) amazonensis infection. Infect Immun. 2003, 71: 3988-3994. 10.1128/IAI.71.7.3988-3994.2003.
Zanin FH, Coelho EA, Tavares CA, Marques-da-Silva EA, Silva Costa MM, Rezende SA, Gazzinelli RT, Fernandes AP: Evaluation of immune responses and protection induced by A2 and nucleoside hydrolase (NH) DNA vaccines against Leishmania chagasi and Leishmania amazonensis experimental infections. Microbes Infect. 2007, 9: 1070-1077. 10.1016/j.micinf.2007.05.012.
Ghosh A, Labrecque S, Matlashewski G: Protection against Leishmania donovani infection by DNA vaccination: increased DNA vaccination efficiency through inhibiting the cellular p53 response. Vaccine. 2001, 19: 3169-3178. 10.1016/S0264-410X(01)00023-8.
Ghosh A, Zhang WW, Matlashewski G: Immunization with A2 protein results in a mixed Th1/Th2 and a humoral response which protects mice against Leishmania donovani infections. Vaccine. 2001, 20: 59-66. 10.1016/S0264-410X(01)00322-X.
Fernandes AP, Costa MM, Coelho EA, Michalick MS, de Freitas E, Melo MN, Luiz Tafuri W, Resende Dde M, Hermont V, Abrantes Cde F, Gazzinelli RT: Protective immunity against challenge with Leishmania (Leishmania) chagasi in beagle dogs vaccinated with recombinant A2 protein. Vaccine. 2008, 26: 5888-5895. 10.1016/j.vaccine.2008.05.095.
Resende DM, Caetano BC, Dutra MS, Penido ML, Abrantes CF, Verly RM, Resende JM, Pilo-Veloso D, Rezende SA, Bruna-Romero O, Fernandes AP, Gazzinelli RT: Epitope mapping and protective immunity elicited by adenovirus expressing the Leishmania amastigote specific A2 antigen: correlation with IFN-gamma and cytolytic activity by CD8+ T cells. Vaccine. 2008, 26: 4585-4593. 10.1016/j.vaccine.2008.05.091.
Mizbani A, Taheri T, Zahedifard F, Taslimi Y, Azizi H, Azadmanesh K, Papadopoulou B, Rafati S: Recombinant Leishmania tarentolae expressing the A2 virulence gene as a novel candidate vaccine against visceral leishmaniasis. Vaccine. 2009, 28: 53-62. 10.1016/j.vaccine.2009.09.114.
Yam KK, Hugentobler F, Pouliot P, Stern AM, Lalande JD, Matlashewski G, Olivier M, Cousineau B: Generation and evaluation of A2-expressing Lactococcus lactis live vaccines against Leishmania donovani in BALB/c mice. J Med Microbiol. 2011, 60: 1248-1260. 10.1099/jmm.0.029959-0.
Rafati S, Gholami E, Hassani N, Ghaemimanesh F, Taslimi Y, Taheri T, Soong L: Leishmania major heat shock protein 70 (HSP70) is not protective in murine models of cutaneous leishmaniasis and stimulates strong humoral responses in cutaneous and visceral leishmaniasis patients. Vaccine. 2007, 25: 4159-4169. 10.1016/j.vaccine.2007.03.006.
Kaur J, Kaur T, Kaur S: Studies on the protective efficacy and immunogenicity of Hsp70 and Hsp83 based vaccine formulations in Leishmania donovani infected BALB/c mice. Acta Trop. 2011, 119: 50-56. 10.1016/j.actatropica.2011.04.007.
Sachdeva R, Banerjea AC, Malla N, Dubey ML: Immunogenicity and efficacy of single antigen Gp63, polytope and polytopeHSP70 DNA vaccines against visceral Leishmaniasis in experimental mouse model. PLoS One. 2009, 4: e7880-10.1371/journal.pone.0007880.
Kaur T, Sobti RC, Kaur S: Cocktail of gp63 and Hsp70 induces protection against Leishmania donovani in BALB/c mice. Parasite Immunol. 2011, 33: 95-103. 10.1111/j.1365-3024.2010.01253.x.
Zhang WW, Charest H, Ghedin E, Matlashewski G: Identification and overexpression of the A2 amastigote-specific protein in Leishmania donovani. Mol Biochem Parasitol. 1996, 78: 79-90. 10.1016/S0166-6851(96)02612-6.
Zhang WW, Matlashewski G: Loss of virulence in Leishmania donovani deficient in an amastigote-specific protein, A2. Proc Natl Acad Sci USA. 1997, 94: 8807-8811. 10.1073/pnas.94.16.8807.
Matlashewski G: Leishmania infection and virulence. Med Microbiol Immunol. 2001, 190: 37-42.
Zhang WW, Matlashewski G: Characterization of the A2-A2rel gene cluster in Leishmania donovani: involvement of A2 in visceralization during infection. Mol Microbiol. 2001, 39: 935-948. 10.1046/j.1365-2958.2001.02286.x.
Zhang WW, Mendez S, Ghosh A, Myler P, Ivens A, Clos J, Sacks DL, Matlashewski G: Comparison of the A2 gene locus in Leishmania donovani and Leishmania major and its control over cutaneous infection. J Biol Chem. 2003, 278: 35508-35515. 10.1074/jbc.M305030200.
Ghedin E, Zhang WW, Charest H, Sundar S, Kenney RT, Matlashewski G: Antibody response against a Leishmania donovani amastigote-stage-specific protein in patients with visceral leishmaniasis. Clin Diagn Lab Immunol. 1997, 4: 530-535.
Srivastava P: Interaction of heat shock proteins with peptides and antigen presenting cells: chaperoning of the innate and adaptive immune responses. Annu Rev Immunol. 2002, 20: 395-425. 10.1146/annurev.immunol.20.100301.064801.
Srivastava P: Roles of heat-shock proteins in innate and adaptive immunity. Nat Rev Immunol. 2002, 2: 185-194. 10.1038/nri749.
Javid B, MacAry PA, Oehlmann W, Singh M, Lehner PJ: Peptides complexed with the protein HSP70 generate efficient human cytolytic T-lymphocyte responses. Biochem Soc Trans. 2004, 32: 622-625. 10.1042/BST0320622.
Li Z: In vitro reconstitution of heat shock protein-peptide complexes for generating peptide-specific vaccines against cancers and infectious diseases. Methods. 2004, 32: 25-28. 10.1016/S1046-2023(03)00183-X.
Hoos A, Levey DL: Vaccination with heat shock protein-peptide complexes: from basic science to clinical applications. Expert Rev Vaccines. 2003, 2: 369-379. 10.1586/147605184.108.40.2069.
Colino J, Snapper CM: Dendritic cells, new tools for vaccination. Microbes Infect. 2003, 5: 311-319. 10.1016/S1286-4579(03)00033-9.
Iniesta V, Carcelen J, Molano I, Peixoto PM, Redondo E, Parra P, Mangas M, Monroy I, Campo ML, Nieto CG, Corraliza I: Arginase I induction during Leishmania major infection mediates the development of disease. Infect Immun. 2005, 73: 6085-6090. 10.1128/IAI.73.9.6085-6090.2005.
Agallou M, Margaroni M, Karagouni E: Cellular vaccination with bone marrow-derived dendritic cells pulsed with a peptide of Leishmania infantum KMP-11 and CpG oligonucleotides induces protection in a murine model of visceral leishmaniasis. Vaccine. 2011, 29: 5053-5064. 10.1016/j.vaccine.2011.04.089.
Wu W, Huang L, Mendez S: A live Leishmania major vaccine containing CpG motifs induces the de novo generation of Th17 cells in C57BL/6 mice. Eur J Immunol. 2010, 40: 2517-2527. 10.1002/eji.201040484.
Korn T, Bettelli E, Oukka M, Kuchroo VK: IL-17 and Th17 Cells. Annu Rev Immunol. 2009, 27: 485-517. 10.1146/annurev.immunol.021908.132710.
Korn T, Oukka M, Kuchroo V, Bettelli E: Th17 cells: effector T cells with inflammatory properties. Semin Immunol. 2007, 19: 362-371. 10.1016/j.smim.2007.10.007.
Fietta P, Delsante G: The effector T helper cell triade. Riv Biol. 2009, 102: 61-74.
Mou Z, Jia P, Kuriakose S, Khadem F, Uzonna JE: Interleukin-17-mediated control of parasitemia in experimental Trypanosoma congolense infection in mice. Infect Immun. 2010, 78: 5271-5279. 10.1128/IAI.00168-10.
This research was supported in part by grant AGL2010-17394 from the Spanish Ministry of Education and Science (MEC) and grant GR58/08 from the Banco Santander-Universidad Complutense. J.C. is an investigator of the “Juan de la Cierva” program (JCI-2009-04069) from the Spanish Ministry of Science and Innovation (MICINN). We thank Dr Manuel Modolell (Max Planck Insitute for Immunbiology, Freiburg, Germany), Dr Antonio Celada (Macrophage Biology Group, Institute of Research in Biomedicine, Barcelona Science Park, Spain) and Dr Fabio M. Cerbán (CIBICI-CONICET, Departamento de Bioquímica Clínica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Argentina) for helpful suggestions and valuable comments to improve detection signal in arginase activity assay. We also thank Dr Alberto Tierrez (Instituto de Investigaciones Biológicas Alberto Sols-CSIC, Madrid, Spain) for his help and advice with transfections.
The authors declare that they have no competing interests.
GDB participated in the design and coordination of the study, and helped to draft the manuscript. PH, JAO, RDF and AHG participated in its design and the discussion section. LOG carried out the detection of HISA70 expression in mammalian cells and participated in the discussion section of this paper. JC conceived the study, carried out the experiments and wrote the paper. All authors read and approved the final manuscript.
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Domínguez-Bernal, G., Horcajo, P., Orden, J.A. et al. Mitigating an undesirable immune response of inherent susceptibility to cutaneous leishmaniosis in a mouse model: the role of the pathoantigenic HISA70 DNA vaccine. Vet Res 43, 59 (2012). https://doi.org/10.1186/1297-9716-43-59
- Parasite Burden
- Arginase Activity
- Major Infection
- Leishmania Parasite
- Neglect Tropical Disease