Effective immunization against cutaneous leishmaniasis with recombinant bacille Calmette-Guérin expressing the Leishmania surface proteinase gp63.

Leishmania parasites cause a spectrum of diseases that afflict the populations of 86 countries in the world. The parasites can survive within the lysosomal compartments of the host's macrophages, unless those macrophages are appropriately activated. Despite the fact that protective immunity can be induced by vaccination with crude parasite preparations, little progress has been made toward a defined vaccine for humans. In this study the gene encoding the Leishmania surface proteinase gp63 was cloned and expressed as a cytoplasmic protein in a bacille Calmette-Guérin (BCG) vaccine strain. BALB/c and CBA/J mice were inoculated with a single dose of recombinant BCG and challenged with infective Leishmania major or Leishmania mexicana promastigotes. Significant protection was observed in both mouse strains against L. mexicana and in CBA/J against L. major, whereas only a delay in L. major growth was seen in BALB/c mice. Recombinant BCG also engendered a strong protective response against challenge with amastigotes of L. mexicana, demonstrating that the induced immune response recognized the intracellular form of the parasite. The results support the view that recombinant BCG expressing gp63 may prove a useful vaccine for inducing protective cell-mediated immune responses to Leishmania species causing American cutaneous leishmaniasis.     

Leishmaniasis in Brazil: XII. Observations on cross-immunity in monkeys and man infected with Leishmania mexicana mexicana, L. m. amazonensis, L. braziliensis braziliensis, L. b. guyanensis and L. b. panamensis.

Cross-immunity trials in monkeys (Cebus apella apella) and observations on experimental and natural infections in man confirm the separate identity of L. mexicana mexicana, L. m. amazonensis, L. b. braziliensis, L. b. guyanensis and L. b. panamensis. Neither L. m. mexicana nor L. m. amazonensis infections gave protection against subsequent challenge with parasites of the L. braziliensis complex; but recovery from infection with subspecies of L. braziliensis in most cases gave firm resistance to infection with the mexicana parasites. The failure of certain New World leishmanias to immunize against each other has an important bearing on taxonomy, future attempts to prepare vaccines against Leishmania, epidemiology and diagnosis.     

Immunity to a salivary protein of a sand fly vector protects against the fatal outcome of visceral leishmaniasis in a hamster model

Visceral leishmaniasis (VL) is a fatal disease for humans, and no vaccine is currently available. Sand fly salivary proteins have been associated with protection against cutaneous leishmaniasis. To test whether vector salivary proteins can protect against VL, a hamster model was developed involving intradermal inoculation in the ears of 100,000 Leishmania infantum chagasi parasites together with Lutzomyia longipalpis saliva to mimic natural transmission by sand flies. Hamsters developed classical signs of VL rapidly, culminating in a fatal outcome 5-6 months postinfection. Saliva had no effect on the course of infection in this model. Immunization with 16 DNA plasmids coding for salivary proteins of Lu. longipalpis resulted in the identification of LJM19, a novel 11-kDa protein, that protected hamsters against the fatal outcome of VL. LJM19-immunized hamsters maintained a low parasite load that correlated with an overall high IFN-gamma/TGF-beta ratio and inducible NOS expression in the spleen and liver up to 5 months postinfection. Importantly, a delayed-type hypersensitivity response with high expression of IFN-gamma was also noted in the skin of LJM19-immunized hamsters 48 h after exposure to uninfected sand fly bites. Induction of IFN-gamma at the site of bite could partly explain the protection observed in the viscera of LJM19-immunized hamsters through direct parasite killing and/or priming of anti-Leishmania immunity. We have shown that immunity to a defined salivary protein (LJM19) confers powerful protection against the fatal outcome of a parasitic disease, which reinforces the concept of using components of arthropod saliva in vaccine strategies against vector-borne diseases.     

Leishmania mexicana and Leishmania major: attenuation of wild-type parasites and vaccination with the attenuated lines

A method for attenuation of Leishmania species by culturing in vitro under gentamicin pressure has been used successfully with Leishmania mexicana, L. major, L. infantum, and L. donovani. The attenuated lines invaded but were unable to survive within bone marrow-derived macrophages in vitro, whereas wild-type parasites survived and multiplied. The attenuated lines of L. mexicana and L. major both failed to induce cutaneous lesions in the majority of BALB/c mice over a minimum 12-week observation period after subcutaneous injection of stationary phase parasites. The attenuated line of L. mexicana retained its properties in gentamicin-free medium over 40 subcultures. The attenuated lines of L. mexicana and L. major both induced significant protection in mice against challenge with wild-type parasites.     

Molecular mass screening to incriminate sand fly vectors of Andean-type cutaneous leishmaniasis in Ecuador and Peru

Sand flies from the Andean areas of Ecuador and Peru were examined for Leishmania infections by using our recently established molecular mass screening method. Leishmanial minicircle DNA-positive sand flies were detected in 3 of 192 and 1 of 462 samples from Ecuador and Peru, respectively. Sand fly species were identified by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of the 18S ribosomal RNA (rRNA) gene, and the positive flies were Lutzomyia (Lu.) ayacuchensis and Lu. peruensis, respectively. Furthermore, cytochrome b and mannose-phosphate isomerase gene sequence analyses identified the parasites from Ecuador and Peru as Leishmania (Leishmania) mexicana and L. (Viannia) peruviana, respectively. Thus, the mass screening method was confirmed to be a powerful tool for sand fly research.     

Antigenic diversity in maxadilan, a salivary protein from the sand fly vector of American visceral leishmaniasis

The salivary protein maxadilan (MAX) is a vasodilator and immunomodulator from the sand fly vector of the protozoan parasite Leishmania chagasi. Vaccinating BALB/c mice with sand fly salivary gland extracts or with MAX protects the host against L. major infection. Because of the potential use of MAX in an anti-Leishmania vaccine, we characterized the vertebrate host IgG response to MAX in the present study. Our immunochemical analysis indicated that antibodies to MAX were detected in BALB/c mice, as well as in pigs and humans, from a area in Nicaragua endemic for Lutzomyia longipalpis. Previous studies demonstrate that the MAX protein is polymorphic on the amino acid level. Our findings suggested that naturally occurring MAX variants were recognized specifically by the host immune system and antigenicity appeared to be associated with amino-acid sequence variability. Thus, antigenic diversity of MAX and possibly of other arthropod salivary proteins may dictate the development of vector-based vaccines(s).     

CTL Responses to DCs Stimulated with Leishmania Antigens Detected by DCs Expressing Leishmania gp63

Background: Leishmania is a pathogenic parasite which infects mononuclear cells in vertebrate hosts. Different strategies have been taken to develop immunity against Leishmania . DCs loaded with immunogenic antigen have resulted in different levels of Th1-type immune response and cytotoxic T lymphocytes (CTL) activity. Objective: To evaluate the potency of DCs primed with soluble Leishmania mexicana antigens (SLA) in developing CTL activity. Methods: DCs were loaded with SLA and injected to Balb/c mice. After two weeks the mice were sacrificed and their splenocytes were used as effector cells in a standard 4-hour cytotoxicity assay against DCs transfected with pcDNA3 containing L. mexicana gp63 gene. Results: Immunization of Balb/c mice with DCs loaded with SLA resulted in high levels of CTL activity against DCs transfected with pcDNA3 containing L. mexicana gp63 gene. Conclusions: The results indicate a high potency for DCs primed with Leishmania antigens in inducing CTL activity, which can be used for developing an immunogenic vaccine against Leishmania.     

Multifarious characterization of leishmania tropica from a Judean desert focus, exposing intraspecific diversity and incriminating phlebotomus sergenti as its vector

The predominant sand fly species collected inside houses in Kfar Adumim, an Israeli village in the Judean Desert that is a focus of cutaneous leishmaniasis, was Phlebotomus papatasi, which was also caught attempting to bite humans. Phlebotomus sergenti, which is rarely seen inside houses, constituted the predominant sand fly species in caves near the village. Leishmania isolates from Ph. sergenti and humans typed as Leishmania tropica. Sand fly and human isolates produced similar small nodular cutaneous lesions in hamsters. Isolates produced excreted factor (EF) of subserotypes A(9) or A(9)B(2), characteristic of L. tropica and reacted with L. tropica-specific monoclonal antibodies. Isoenzyme analysis consigned the strains to the L. tropica zymodemes MON-137 and MON-275. Molecular genetic analyses confirmed the strains were L. tropica and intraspecific microheterogeneity was observed. Genomic fingerprinting using a mini-satellite probe separated the L. tropica strains into two clusters that were not entirely congruent with geographic distribution. These results support the heterogeneous nature of L. tropica and incriminate Ph. sergenti as its vector in this Judean Desert focus.     

Detection and identification of Leishmania species within naturally infected sand flies in the andean areas of ecuador by a polymerase chain reaction

The surveillance of prevalent Leishmania and sand fly species in endemic areas is important for prediction of the risk and expansion of leishmaniasis. In this study, we developed a polymerase chain reaction (PCR)-based method for detection of Leishmania minicircle DNA within individual sand flies. Using this method, we detected minicircle DNA in 6 (3.3%) of 183 sand flies, while 5 (3.5%) of 143 were positive for Leishmania promastigotes in the same areas by microscopic examination. The species were identified as Leishmania (Leishmania) mexicana by nucleotide sequencing of the cytochrome b gene. Additionally, all the Leishmania-positive sand flies were identified as Lutzomyia ayacuchensis by the restriction enzyme digestion of the PCR-amplified 18S ribosomal RNA gene fragments. Since this combined method is relatively easy and can process a large number of samples, it will be a powerful tool for the rapid identification of prevalent sand fly and Leishmania species as well as monitoring the infection rate in sand fly populations in endemic areas.     

Cutaneous leishmaniasis following local trauma: a clinical pearl.

Cutaneous leishmaniasis is acquired from the bite of an infected sand fly and can result in chronic skin lesions that develop within weeks to months after a bite. Local trauma has been implicated as a precipitating event in the development of skin lesions in patients who have been infected with Leishmania species. Here we report a case series and review the literature on patients who developed cutaneous leishmaniasis after local trauma, which may familiarize clinicians with this presentation.     

Protection against cutaneous leishmaniasis in outbred vervet monkeys using a recombinant histone H1 antigen

Infection with Leishmania major parasites results in the development of cutaneous ulcerative lesions on the skin. We investigated the protective potential of a single, recombinant histone H1 antigen against cutaneous leishmaniasis in an outbred population of vervet monkeys, using Montanide adjuvant. Protection was assessed by challenging the animals with a mixture of vector sand fly salivary-gland lysate and a low dose of in vitro-derived parasites, thus more closely mimicking natural infection induced by L. major. The course of infection in immunized monkeys was compared with that of animals that had healed from a primary infection and were immune. The monkeys immunized with recombinant histone H1 showed a reduced development of lesion size, compared with controls. Our study therefore illustrates the potential use of histone H1 as a vaccine candidate against cutaneous leishmaniasis in humans.     

CTL responses to Leishmania mexicana gp63-cDNA vaccine in a murine model

Immunity to Leishmania is believed to be strongly dependent upon the activation of Th1 immune responses, although the exact role of cytotoxic T lymphocytes (CTLs) has not yet been determined. The aims of this study were to establish a suitable cytotoxicity assay to measure CTL activity and to compare immunity induced by Leishmania mexicana gp63 cDNA via i.m. injection and gene gun immunization in the BALB/c mouse model. The CTL activity was evaluated by short-term ⁵¹Cr-release cytotoxicity assays against CT26 tumour cells transfected with L. mexicana gp63 cDNA and dendritic cells (DCs) loaded with soluble Leishmania antigen (SLA) as targets. The results clearly demonstrated that higher protection to L. mexicana infection was induced by gene gun DNA-immunization vs. i.m. injection. Cytotoxic T lymphocyte activity of splenocytes was observed in mice immunized either with L. mexicana gp63 cDNA or SLA and long-lived CTL activity was observed in immunized and/or re-challenged mice but not naïve mice infected with the parasite.     

Sand fly feeding on noxious plants: a potential method for the control of leishmaniasis.

The sand fly Phlebotomus papatasi transmits Leishmania major, which causes cutaneous leishmaniasis, in vast regions of the Old World. In addition to blood, the sand flies feed on plants. In a study of this diet, we observed that one night of feeding on branches of Solanum jasminoides, Ricinus communis, or Bougainvillea glabra drastically shortened the life span of the sand flies. Flowering B. glabra attracted P. papatasi in the field. Nevertheless, in the region endemic for L. major in yards abounding with vector sand flies, the number of P. papatasi trapped near hedges of B. glabra was eight times less (62 versus 502 flies trapped) than in the control sites. The results imply that B. glabra affords local protection against sand fly bites and decreases the risk of leishmaniasis. We suggest that this and other ornamental plants that are harmful to sand flies can be used as a tool for this purpose.     

DNA vaccination with KMP11 and Lutzomyia longipalpis salivary protein protects hamsters against visceral leishmaniasis

It was recently shown that immunization of hamsters with DNA plasmids coding LJM19, a sand fly salivary protein, partially protected against a challenge with Leishmania chagasi, whereas immunization with KMP11 DNA plasmid, a Leishmania antigen, induced protection against L. donovani infection. In the present study, we evaluated the protective effect of immunization with both LJM19 and KMP11 DNA plasmid together. Concerning the protection against an infection by L. chagasi, immunization with DNA plasmids coding LJM19 or KMP11, as well as with both plasmids combined, induced IFN-γ production in draining lymph nodes at 7, 14 and 21 days post-immunization. Immunized hamsters challenged with L. chagasi plus Salivary Gland Sonicate (SGS) from Lutzomyia longipalpis showed an enhancement of IFN-γ/IL-10 and IFN-γ/TGF-β in draining lymph nodes after 7 and 14 days of infection. Two and five months after challenge, immunized animals showed reduced parasite load in the liver and spleen, as well as increased IFN-γ/IL-10 and IFN-γ/TGF-β ratios in the spleen. Furthermore, immunized animals remained with a normal hematological profile even five months after the challenge, whereas L. chagasi in unimmunized hamsters lead to a significant anemia. The protection observed with LJM19 or KMP11 DNA plasmids used alone was very similar to the protection obtained by the combination of both plasmids.     

Cross-immunity experiments between different species or strains of Leishmania in rhesus macaques (Macaca mulatta)

This study evaluates cross-immunity in rhesus monkeys (Macaca mulatta) previously infected with one species of Leishmania and have had self-cured disease or were cured by antimony-based therapy upon development of full-blown disease. We found that a self-healing cutaneous leishmaniasis (CL) following experimental infection with Leishmania (Leishmania) major induces significant protection for L. (L.) amazonensis and L. (Viannia) guyanensis, and was dependent on time of re-challenge by L (L.) amazonensis after animals had recovered from primary lesions, but lacked protection against L. (V.) braziliensis. In contrast, monkeys that recovered from L. (V.) braziliensis CL or L. (L.) chagasi visceral leishmaniasis following chemotherapeutic intervention were protected by challenge with L. (V.) braziliensis and L (L.) amazonensis. These findings indicate the relative variability in protection after self-cure or drug-cured experimental leishmaniasis to challenge by heterologous leishmanial parasites. Further studying the immune response may provide information regarding relevant factors influencing cross-protective immunity.     

热休克蛋白70（hsp70）基因对利什曼原虫中国分离株的系统发育分析

目的选用hsp70基因探讨我国各疫区不同宿主利什曼原虫的种系发育关系。方法采用PCR方法扩增分离自中国各疫区不同宿主的利什曼原虫之热休克蛋白70（hsp70）基因,将扩增的目的片段纯化后测序。使用MEGA5．0软件对测序产物进行多序列比对并构建系统发育树,同时与国外利什曼原虫分离株的hsp70序列共同分析,观察利什曼原虫中国分离株在世界范围利什曼原虫的系统发育和分子进化中的地位。结果中国各流行疫区利什曼原虫均获得约1300bp长度的hsp70基因片段,存在丰富的多态位点。中国23株利什曼原虫经hsp70基因分型聚集为四群,其中人来源的原虫分离株均在A群,为杜氏利什曼原虫（L．donovani）,并分为杜氏利什曼原虫指名亚种（L.donovani）和杜氏利什曼原虫婴儿亚种（L．infantum）两个亚支;分离自新疆沙鼠或白蛉的6株利什曼原虫聚在B群,为都兰利什曼原虫（L．turanica）;分离自新疆白蛉和内蒙古沙鼠的两株利什曼原虫为C群沙鼠利什曼原虫（L．gerbilli）;分离自内蒙古蜥蜴和新疆白蛉的两株原虫为D群蜥蜴利什曼原虫（Sauroleishmania）。结论利什曼原虫中国分离株属于利什曼原虫亚属的不同种和蜥蜴利什曼原虫亚属,我国利什曼原虫分离株的种系发育复杂多样。     

Influence of vertebrate blood meals on the development of Leishmania (Viannia) braziliensis and Leishmania (Leishmania) amazonensis in the sand fly Lutzomyia migonei (Diptera: Psychodidae)

The effect of blood meals from humans and seven domestic, wild, or laboratory animals (dogs, horses, chickens, rats, opossums, mice, and hamsters) on the development of Leishmania braziliensis and L. amazonensis was studied in the sand fly Lutzomyia migonei. The development of L. braziliensis and L. amazonensis exhibited peripylarian and suprapylarian patterns of development, respectively, in the sand fly gut with all blood meals tested. The blood meal sources influenced the infection rate of the sand flies. In both the Leishmania species, the highest parasite density was obtained with blood from wild rats followed by skunk, human, and horse. The epidemiological significance of these observations may be related to the distribution of leishmaniasis and needs to be evaluated further.     

The correlation between delayed hypersensitivity, lymphocyte activation and protective immunity in experimental murine leishmaniasis

The growth of Leishmania major and Leishmania mexicana lesions and the concomitant development of delayed-type hypersensitivity (DTH) to homologous or heterologous soluble antigen was studied in BALB/c and CBA/Ca mice. Although CBA/Ca mice are highly susceptible to L. mexicana, developing non-healing lesions, they are resistant to L. major; while BALB/c mice develop non-healing lesions when infected with either species. The development of resistance was associated with the acquisition of DTH which peaked at 48 h (L. major infected CBA/Ca mice). Non healing lesions were associated with either negative DTH (L. major infected BALB/c mice) or DTH that peaked at 24 h but had significantly subsided by 48 h (L. mexicana infected CBA/Ca and BALB/c mice). The latter response was associated with basophilic infiltration of the skin test site. Pre-irradiating (600 rad) CBA/Ca and BALB/c mice induced resistance against L. mexicana and L. major respectively in conjunction with the appearance of 48 h DTH to the homologous antigen. There was clear dissociation in the skin reactivity produced by the heterologous antigen. Thus L. major-derived antigen failed to produce DTH in L. mexicana infected mice of either strain. L. mexicana-derived antigen on the other hand produced a quicker response and of greater magnitude than the homologous antigen in L. major infected CBA/Ca mice. This correlated well with the strong cross-immunity induced by L. major in these mice to L. mexicana infection.     

Leishmania amazonensis infections in Oryzomys acritus and Oryzomys nitidus from Bolivia

Three of thirteen Oryzomys acritus, Emmons and Patton 2005 (Rodentia: Muridae: Sigmodontinae) and 3 of 17 Oryzomys nitidus, Thomas 1884, collected from No?l Kempff National Park, Bolivia, from 2002 to 2005, tested positive for Leishmania (Leishmania) amazonensis or L. (L.) mexicana and negative for Leishmania (Viannia) spp. using the polymerase chain reaction (PCR). Based on previous records of L. (L.) amazonensis in humans, rodents, and sand flies from Bolivia, and the geographic distributions of L. (L.) amazonensis and L. (L.) mexicana, it was concluded that the Oryzomys were infected with L. (L.) amazonensis. These results identify two additional species of Oryzomys as hosts of L. (L.) amazonensis, and identify an ecological region of Bolivia where L. (L.) amazonensis is enzootic.