Short report: occurrence of Leishmania donovani DNA in donated blood from seroreactive Brazilian blood donors.

Human visceral leishmaniasis (kala-azar) transmitted by blood transfusion has been described in previous reports. Seroprevalence of antibodies to Leishmania donovani was shown to be related to prior blood transfusions in multiply transfused hemodialysis patients in Natal, Rio Grande do Norte, Brazil. In this study, a possible correlation between seroreactivity and the presence of L. donovani DNA was investigated in asymptomatic healthy blood donors. Sera were tested using the fucose mannose ligand (FML) ELISA, which was shown to have a sensitivity of 100%, a specificity of 96-100%, reliability, and diagnostic and prognostic potential for the detection of human and canine kala-azar, respectively. Leishmanial DNA was assessed by the polymerase chain reaction (PCR) and dot-blot hybridization techniques in blood and bone marrow samples. Among 21 FML-seroreactive asymptomatic blood donors, 5 (24%) were positive by the PCR and 9 (43%) were positive in a dot-blot assay of blood samples, showing a significant correlation (chi2 = 14.24, P < 0.01). No Leishmania DNA was detected in 20 FML non-reactive blood donors. Our results point to the need for control of transmission of kala-azar by blood transfusion in areas endemic for this disease.

The fucose-mannose ligand-ELISA in the diagnosis and prognosis of canine visceral leishmaniasis in Brazil.

The fucose-mannose ligand (FML)-ELISA assay showed a sensitivity of 100% and a specificity of 100% in diagnosis of canine visceral leishmaniasis (CVL) (kala-azar) in sera from naturally infected dogs from São Gonçalo do Amaranto, Rio Grande de Norte, Brazil. The overall prevalence of antibodies to Leishmania in the endemic area was 23% (79 of 343). Seroreactivity detected by a Leishmania chagasi immunofluorescent (IF) assay was much lower (2.9%) and similar to the percentage of dogs with kala-azar symptoms (2.6%). Twenty-one of 21 asymptomatic, FML-seropositive animals died of kala-azar in a period ranging from 0 to 6 months after diagnosis. The predictive value was 100% for the FML-ELISA, 43% for an L. mexicana ELISA, and 24% for the L. mexicana and L. chagasi IF assays, respectively. In experimentally infected dogs, all assays detected seropositivity between 90 and 120 days after infection. Since the current strategy for control of CVL is based on detection and destruction of infected dogs, the highly predictive, sensitive, and specific FML-ELISA represents a useful tool for field control of the disease.

Prevalence of anti-Leishmania donovani antibody among Brazilian blood donors and multiply transfused hemodialysis patients.

The prevalence of anti-Leishmania donovani antibodies was investigated in 1,500 Brazilian blood donors and multiply transfused hemodialysis patients. Sera were tested using the fucose-mannose ligand (FML) ELISA, which was shown to have 100% sensitivity and 96% specificity for kala-azar. Among 1,194 volunteer blood donors, seroreactivity was 9%, increasing to 25% in a periurban kala-azar focus. However, higher positivity (37%) was found in multiply transfused hemodialysis patients from Natal, where kala-azar is constantly present in low numbers (endemic), with sporadic outbreaks in localized regions (endemic and epidemic). Risk factors included blood transfusion, which was significantly associated with the presence of anti-Leishmania antibodies (chi2 = 8.567, P < 0.005), but did not include potential exposure to sandfly bites (chi2 = 0.033, P > 0.1). The prevalence significantly decreased to 7% in hemodialysis patients from Rio de Janeiro, where kala-azar is only occasionally seen, and was 0% in patients undergoing continuous ambulatorial peritoneal dialysis. The prospective analysis of 27 FML-seroreactive donors from Natal revealed amastigotes of Leishmania in the bone marrow of one subject while four had clinical complaints, including splenomegaly and hepatosplenomegaly. Our results point to the need for control of blood transfusion as a possible route for transmission of kala-azar in endemic areas.

Impact of canine control on the epidemiology of canine and human visceral leishmaniasis in Brazil.

Brazil is the only country endemic for zoonotic visceral leishmaniasis (ZVL) that regularly conducts epidemiologic and prophylactic control programs that involve the treatment of human cases, insect vector control, and the removal of seropositive infected dogs. This report reviews 60 studies reporting data on the efficacy of these recommended control tools and concludes that in Brazil 1) eradication of the disease in Minas Gerais was achieved by the concomitant use of the three control methods, 2) although seropositivity by an immunofluorescent assay is not completely related to infectiousness, the removal of seropositive dogs leads to a significant reduction of canine and human incidence, 3) improvement of the sensitivity of the diagnostic tool used for canine control should optimize the efficacy of control, and 4) although difficult and expensive, the public health dog control campaigns performed in Brazil reduced the incidence of ZVL and should be maintained since treatment of dogs is an unrealistic intervention, both because of its prohibitive cost and relatively poor effectiveness.

Leishmania donovani: titration of antibodies to the fucose-mannose ligand as an aid in diagnosis and prognosis of visceral leishmaniasis.

The fucose-mannose ligand (FML) is a complex glycoprotein fraction present on promastigotes and amastigotes of Leishmania donovani. It participates in parasite interaction with host macrophages in a species-specific pattern. We have tested its use in immunodiagnosis of visceral leishmaniasis (VL) in a recent outbreak in Rio Grande do Norte, north-east Brazil. Enzyme-linked immunosorbent assay (ELISA) of low concentrations of FML in 462 sera showed 100% sensitivity and 96% specificity. The FML-ELISA identified patients with overt VL (P < 0.001, compared to normal sera). It could also identify inhabitants of the endemic area who had incipient or subclinical infection with potentially severe clinical disease: more than 20% of apparently healthy subjects with a positive ELISA for FML developed overt VL during the following 10 months. FML-ELISA reactivity decreased in all patients during treatment, and became negative after parasitological cure. No cross-reaction was observed in patients infected with other Leishmania species, nor in those with Chagas disease. Determination of antibody response to FML may be useful in diagnosis of VL and in identifying patients without overt disease but with a high risk of developing severe VL.

Leishmania donovani surface glycoconjugate GP36 is the major immunogen component of the fucose-mannose ligand (FML).

Leishmania donovani promastigote glycoconjugate ligands, studied in our laboratory, that interact with the internalization receptors on BALB/c macrophages: the 'fucose mannose ligand' (FML), the 'phosphate mannogalactan ligand' (PMGL), and the 'lipopeptidephosphoglycan' (LPPD), interfered also with interaction between amastigotes and host cells in vitro. Among the three compounds studied, the FML was shown to be the most potent inhibitor of both promastigote and amastigote internalization, and to be present on parasite surface during the vertebrate-host cycle. The FML, but not the other two glycoconjugates, is a potent immunogen in rabbits (ELISA, agglutination and immuno-blots). Rabbit hyperimmune sera recognized essentially the 36 kDa band of FML. Mouse monoclonal antibodies against FML recognized either the 36 kDa or the 55 kDa band. No cross-reactivity between these two FML components was detected. No antigenic similarity could be detected between the 36 and 55 kDa bands of FML and the 'GP63' (promastigote surface proteinase) major surface leishmanial antigen. The 36 kDa-glycoprotein was identified as the major FML antigenic fraction and designated 'GP36'. The integrity of the glycidic moiety was necessary for its antigenicity. This L. donovani surface glycoprotein is apparently one of the major molecules involved in interactions between the parasite and the vertebrate host.

Occurrence of Leishmania donovani parasitemia in plasma of infected hamsters.

Intracardiac transfusion of plasma, mononuclear cell fraction and blood of infected hamster donors induced visceral leishmaniasis in normal hamster receptors. At the moment of transfusion, the donors already showed all the typical signs of the disease: ascites, cachexia, as well as splenomegaly and a high parasite load in the spleen and liver. All transfused hamsters developed typical visceral leishmaniasis between 90 and 120 days, indicating that all blood products were infectious. Transfusion of the mononuclear cell fraction induced the highest values of parasitic load (spleen, 766 Leishman Donovan Units (LDU); liver, 2650 LDU), splenomegaly and hepatomegaly (spleen-liver/body relative weight: 1.130 and 6.870, respectively). Animals that received the plasma fraction also developed visceral leishmaniasis, showing similar parasitic load (spleen, 107 LDU; liver, 220 LDU) and spleen-liver/body relative weight (1.005 and 6.35, respectively) than those transfused with whole blood. The finding of typical Leishmania donovani infection in animals transfused with plasma demonstrates the possibility of the extracellular location of parasites, free in this blood fraction deprived of red and white blood cells. Fluorescence-assisted cell sorter analysis (FACS) of plasma showed the presence of particles corresponding in size to amastigotes, which fluoresced strongly with the serum of a patient with Kala-azar (73%), but not with normal serum.

Experimental murine Leishmania donovani infection: immunoprotection by the fucose-mannose ligand (FML).

The fucose-mannose ligand (FML) of Leishmania donovani is a complex glycoprotein fraction present in pro- and amastigotes, that interferes with parasite-macrophage interactions in vitro. In the present study, we have tested the potential immunoprotective effect of FML on L. donovani infection in inbred female BALB/c mice. The protection schemes included three weekly intraperitoneal administrations of FML, supplemented or not with saponin. Mice were challenged by intravenous injections of 2 x 10(7) amastigotes of Leishmania donovani (LD-1S/MHOM/SD/00-strain 1S) obtained from CB hamsters' infected spleens. After 15 days of infection, we monitored the splenocyte proliferative response to FML in vitro by ELISA for specific antibody response, and by parasite quantification as "Leishman-Donovan Units" in liver. A significant (P < 0.001) protective effect of FML with saponin, but not of FML or saponin alone, was shown by the reduction of parasite burden in liver and by the enhancement of splenocyte proliferation. The antibody response, very low at 15 days of infection in both untreated and control animals, showed a pronounced increase (P < 0.001) in animals sensitized with FML/saponin. Taken together, our results represent a 79.1 and 89.1% increase in specific proliferative and antibody responses, respectively, and an 84.4% protection in reduction of parasite liver burden. The protective potential was specifically due to FML (P < 0.001). Under the present conditions, no toxic or nonspecific effect could be attributed to saponin. A detailed study of the molecular events related to vaccination against murine visceral leishmaniasis with total and fractionated FML is currently underway.

An improved method for the isolation of erythrocyte antigen band-3.

An improved method for isolation of human and Rhesus monkey band-3 separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is described. Purified band-3 was obtained from human hemoglobin-free ghosts (Hb-free ghosts) after SDS-PAGE by chemical elution+sonication (CE+S). The section of the gel corresponding to the antigen was cut out, mechanically disrupted and incubated in 1% NaHCO3 containing 1% SDS, for 2 h, with shaking, at room temperature, followed by overnight incubation at 4 degrees C. The preparation was subsequently sonicated and clarified by centrifugation. Supernatants were dialyzed against distilled water, their protein contents were measured, and the presence of purified band-3 was demonstrated by SDS-PAGE. A calibration curve was developed for assay of CE+S material using densitometric evaluation of the protein profile on SDS-PAGE. An amount of 37.5 mg of Hb-free ghosts gave 3.15 mg of purified band-3 after CE+S, corresponding to an 8.4% yield. Rabbits were immunized with 50 micrograms CE+S antigen. Sera were collected and assayed by Western blot analysis against its proteolytic fragments, which were obtained from packed red blood cells by treatment with protease type VI from Streptomyces griseus (1 h at 37 degrees C), followed by extensive washing and hypotonic lysis. Specific antibodies recognized band-3 and its proteolytic fragments 60 and 63 kDa in human ghosts obtained from different blood donors, confirming the genetic polymorphism. Analogous serum obtained against the Rhesus monkey band-3 proteolytic fragment 63 kDa recognized the human antigen and its respective fragments. These results indicate the existence of similarities between these two species of band-3, suggesting the potential use of this technique in taxonomic and phylogenetic studies. Purification by CE+S is an efficient and rapid method for isolation of band-3 and its fragments with satisfactory yields and maintenance of both their immunogenic and antigenic properties.

Transmission of visceral leishmaniasis by blood transfusion in hamsters.

We have studied the transmission of visceral leishmaniasis by blood transfusion in the CB hamster model. Five normal CB hamsters (females, 2.5 months old) received a 0.1-ml blood transfusion from a donor that had been infected with 10(7) amastigotes of Leishmania donovani 90 days prior to the blood harvest. The development of the disease in transfused animals was monitored by the increase in anti-Leishmania serum antibodies, splenomegaly, and spleen and liver parasitic burdens. The transfused hamsters developed all the typical signs of the disease, i.e., ascites, cachexia and death. The scores of anti-Leishmania antibodies (1.345) and the level of parasite load (spleen Leishman Donovan units of Stauber (LDU) = 471, liver LDU = 378) in transfuse hamsters were similar to those observed in hamsters experimentally infected with 10(7) amastigotes (P > 0.05, Student t-test). Our results demonstrate that blood transfusion is an effective route for transmission of visceral leishmaniasis, and we point out that adequate precautions should be taken at blood banks in the regions where leishmaniasis is endemic.

The FML (Fucose Mannose Ligand) of Leishmania donovani. a new tool in diagnosis, prognosis, transfusional control and vaccination against human kala-azar.

The Fucose-Mannose Ligand (FML) of Leishmania donovani is a complex glycoproteic fraction. Its potential use as a tool for diagnosis of human visceral leishmaniasis was tested with human sera from Natal, Rio Grande do Norte, Brazil. The FML-ELISA test, showed 100% sensitivity and 96% specificity, identifying patients with overt kala-azar (p < 0.001, when compared to normal sera), and subjects with subclinical infection. More than 20% apparently healthy subjects with positive reaction to FML developed overt kala-azar during the following 10 months. In the screening of human blood donnors, a prevalence of 5% of sororeactive subjects was detected, attaining 17% in a single day. The GP36 glycoprotein of FHL is specifically reconized by human kala-azar sera. The immunoprotective effect of FML on experimental L. donovani infection was tested in swiss albino mice. The protection scheemes included three weekly doses of FML, supplemented or not with saponin by the subcutaneous or intraperitoneal routes and challenge with 2 x 10(7) amastigotes of Leishmania donovani. An enhancement of 80.0% in antibody response (p < 0.001) and reduction of 85.5% parasite liver burden (p < 0.001) was detected in animals immunized with FML saponin, unrespectively of the immunization route.

Immunotherapy with the saponin enriched-Leishmune vaccine versus immunochemotherapy in dogs with natural canine visceral leishmaniasis.

Leishmune, the first licensed vaccine for prophylaxis against canine visceral leishmaniasis (CVL) and is also immunotherapeutic when used with double saponin adjuvant concentration. The Leishmune therapeutic vaccine was assessed for immunotherapy (IT) in 31 infected dogs and for immunochemotherapy (ICT) in combination with allopurinol or amphotericinB/allopurinol, in 35 dogs. Compared to infected untreated control dogs, at month 3, both treatments increased the proportion of dogs showing intradermal response to Leishmania antigen to a similar extent (from 8 to 67%, in the IT and to 76%, in the ICT groups), and conversely reduced from 100 to 38% (IT) and to 18% (ICT) the proportion of symptomatic cases, from 54 to 12% (IT) and to 15% (ICT) the proportion of parasite evidence in lymph nodes and from 48 to 19% (IT) and 12% (ICT) the proportion of deaths, indicating that the immunotherapy with enriched-Leishmune vaccine promotes the control of the clinical and parasitological signs of CVL rendering most dogs asymptomatic although PCR positive. By month 8, negative lymph node PCR results were obtained in 80% of the ICT-treated dogs, but only in 33% of the IT group (p=0.0253), suggesting that the combination of additional chemotherapy with Leishmune-enriched saponin vaccination abolished, not only the symptoms but also the latent infection condition, curing the dogs. The animals were followed up until 4.5 years after the beginning of the experiment and, compared to the untreated control group at month 3 (12/25 dogs; 48%), a decrease in the rate of CVL deaths was only seen after ICT treatment (7/35 dogs; 20%; 0.0273) but not after IT treatment (10/31 dogs; 32%; p=0.278), pointing out an additional advantage of the ICT treatment with the enriched-Leishmune in the control and cure of CVL.

Nucleoside hydrolase DNA vaccine against canine visceral leishmaniasis.

The Nucleoside Hydrolase (NH36) is the main marker of the FML complex of Leishmania donovani, antigen of the licensed Leishmune® vaccine for prophylaxis of canine visceral leishmaniasis. As a DNA vaccine in mice, it induces a TH1 immune response. We vaccinated mongrel dogs with the VR1012NH36 vaccine for prophylaxis and immunotherapy against a high dose Leishmania chagasi infection (7 x 108 infective amastigotes). The untreated controls developed more symptoms, higher parasite/lymphocyte ratio, smaller DTH reactions, lower proportions of NH36-specific CD4+ cells and sustained NH36-specific CD8+ cell counts than dogs of the prophylaxis group. In the immunotherapy treated group, enlarged DTH reactions, enhanced CD4+ and sustained CD8+ lymphocyte proportions were also detected, however, without reduction of symptoms or parasite/lymphocyte ratio, indicating that the vaccine was sufficiently potent to prevent but not to control the disease. Both treatments determined higher survival rates. Anti-FML antibodies increased in vaccinated and control dogs while anti-NH36 antibodies were only increased in vaccinees (p= 0.000). The parasite load of an untreated survivor control dog (638.05 parasites) felt outside the IC95% of that of vaccinated dogs (32.02, IC95% 9.45-64.59) suggesting that both vaccination treatments succeeded in reducing the Leishmania infective burden. Accordingly, an untreated control dog showed lower levels of IFN γ-ß, IL-2, IL4 but not IL-10 ß actin-relative quantification. We conclude that the VR1012-NH36 vaccine induces strong prophylactic protection and a milder immunotherapeutic effect against a high dose canine experimental infection with Leishmania chagasi.

Immunogenicity assay of the Leishmune vaccine against canine visceral leishmaniasis in Brazil.

Leishmune is the industrialized version of the FML-saponin vaccine which has been shown to develop 92-95% protection in vaccinated dogs and 76-80% vaccine efficacy against field canine visceral leishmaniasis (CVL) in Brazil. Leishmune has been proven to be safe and tolerable and a transmission-blocking vaccine which renders vaccinated dogs non-infectious to sand fly vectors. In the present investigation, 550 healthy seronegative dogs of endemic and epidemic areas of Brazil were monitored for Leishmune-induced immunogenicity during a 2-year trial. Another group of 588 untreated exposed dogs was also studied in parallel. Both groups were seronegative on day 0. The strong immunogenicity induced by Leishmune vaccine was demonstrated by the 98% of FML-seroconversion, increase in absorbencies, the 82.7% DTH positive reactions and increase in skin test size diameters, the average increase in CD8+ total lymphocytes population in blood (27.1%), expected for QS21 saponin-containing vaccine, the sustained proportions of CD4+ T cells, and the average increased proportions of CD21+ B lymphocytes (42.3%). The Leishmune-induced protection against CVL is demonstrated by the results: 98.8% asymptomatic dogs (at the end of first year) and 99% healthy survivors (at the end of the second year) among vaccinated dogs, compared to the 79.4% asymptomatic and 61% survivor dogs (p<0.001) monitored in the untreated exposed cohort. In spite of the low vaccine coverage, it was possible to detect a 66.1% (p<0.005) reduction in Belo Horizonte and an 80.2% (p<0.005) reduction in Araçatuba of the incidence of CVL among vaccinated dogs, when compared to the global incidence of CVL of each town, respectively. Our preliminary results support the potential use of Leishmune to prevent CVL epidemics.

Assessment of the monoterpene, glycidic and triterpene-moieties' contributions to the adjuvant function of the CP05 saponin of Calliandra pulcherrima Benth during vaccination against experimental visceral leishmaniasis.

The CP05 saponin from Calliandra pulcherrima Benth, shows remarkable similarities to the QS21 saponin of Quillaja saponaria Molina. Both shared a monoterpene hydrophobic moiety, a glycidic chain attached to the triterpene C28, and three sugars attached to C3. Different from QS21, the CP05 does not show the aldehyde group in triterpene C4 involved in TH1 response. Balb/c mice were immunized either intact saponin (CP05), the monoterpene-deprived (BS), the C28 carbohydrate-deprived (HS) or the sapogenin fraction, in formulation with the FML antigen of Leishmania donovani and challenged with 2 x 10(8) amastigotes of L. chagasi. While the CP05 induced 90% survival and 92.1% parasite reduction, a 100% survival and 94.1% protection were detected after the BS-vaccine treatment, indicating that the monoterpene acylated moiety, absent in the BS vaccine, is not necessary for the induction of a protective global TH1 response. Only the DTH response of BS vaccines was mildly lower than that of CP05 vaccinees. Maximal anti-FML antibody, CD4(+) and CD8(+) Leishmania specific lymphocytes, IFN-gamma splenocyte secretion, reduction in parasite load and survival was also detected for the BS vaccine. The HSFML vaccine showed diminished responses in all tested variables, except for IFN-gamma secretion, indicating that the integrity of the carbohydrate moiety attached to C28 is mandatory for the these functions. No protection was induced by the sapogenin-FML indicating that the CP05 triterpene which lacks the C4 aldehyde group, is not a immunostimulating compound. No contribution to protection was detected in the CP05 saponin treated control group supporting the specificity of the FML antigenic preparation.

Safety trial using the Leishmune vaccine against canine visceral leishmaniasis in Brazil.

A group of 600 healthy and asymptomatic dogs from Brazilian canine visceral leishmaniasis endemic areas was vaccinated with three sc doses of Leishmune which is the industrialized formulation of the FML-saponin, recently licensed for commercialization in Brazil, which previously showed 76-80% vaccine efficacy against canine visceral leishmaniasis. Safety evaluation was performed for 14 days after each vaccine injection and disclosed transient reactions of local pain (40.87%), anorexia (20.48%), apathy (24.17%), local swelling reactions (15.90%), vomit (2.4%) and diarrhoea (1.5%). All effects showed significantly correlating declines, from the first to the third dose (p<0.0001). Most of the noticed reactions of pain (73%), anorexia (79%) and local swelling (84.7%) were mild. No significant differences between puppies and adults dogs were found in the number of adverse reactions. Adult dogs developed however, 94.5% of the small swelling reactions (<3 cm), and indicating that they are more resistant to the inflammatory response promoted by the saponins. No dead by anaphylaxis occurred, and only two dogs (0.1%) showed allergic reactions (facial oedema and itching) after the third dose. Transient alopecia on injection site occurred in only five poodles (0.28%) with total recovery and no need of treatment. All the mild adverse events in response to Leishmune injection were transient and disappeared before the injection of the following vaccine dose, confirming the tolerability of the vaccine. The Leishmune preparation was less haemolytic (HD(50)=180 microg/ml) than expected for a QS21 saponin-containing vaccine, indicating that its formulation with the FML antigen diminished the potential in vitro toxicity.

Immunotherapy against experimental canine visceral leishmaniasis with the saponin enriched-Leishmune vaccine.

In order to assess the immunotherapeutic potential on canine visceral leishmaniasis of the Leishmune vaccine, formulated with an increased adjuvant concentration (1mg of saponin rather than 0.5mg), 24 mongrel dogs were infected with Leishmania (L.) chagasi. The enriched-Leishmune vaccine was injected on month 6, 7 and 8 after infection, when animals were seropositive and symptomatic. The control group were injected with a saline solution. Leishmune-treated dogs showed significantly higher levels of anti-FML IgG antibodies (ANOVA; p<0.0001), a higher and stable IgG2 and a decreasing IgG1 response, pointing to a TH1 T cell mediated response. The vaccine had the following effects: it led to more positive delayed type hypersensitivity reactions against Leishmania lysate in vaccinated dogs (75%) than in controls (50%), to a decreased average of CD4+ Leishmania-specific lymphocytes in saline controls (32.13%) that fell outside the 95% confidence interval of the vaccinees (41.62%, CI95% 43.93-49.80) and an increased average of the clinical scores from the saline controls (17.83) that falls outside the 95% confidence interval for the Leishmune immunotherapy-treated dogs (15.75, CI95% 13.97-17.53). All dogs that received the vaccine were clustered, and showed lower clinical scores and normal CD4+ counts, whereas 42% of the untreated dogs showed very diminished CD4+ and higher clinical score. The increase in clinical signs of the saline treated group was correlated with an increase in anti-FML antibodies (p<0.0001), the parasitological evidence (p=0.038) and a decrease in Leishmania-specific CD4+ lymphocyte proportions (p=0.035). These results confirm the immunotherapeutic potential of the enriched-Leishmune vaccine. The vaccine reduced the clinical symptoms and evidence of parasite, modulating the outcome of the infection and the dog's potential infectiosity to phlebotomines. The enriched-Leishmune vaccine was subjected to a safety analysis and found to be well tolerated and safe.

Immunotherapy against visceral leishmaniasis with the nucleoside hydrolase-DNA vaccine of Leishmania donovani.

The nucleoside hydrolase (NH36) of Leishmania (L.) donovani is a vital enzyme which releases purines or pyrimidines of foreign DNA to be used in the synthesis of parasite DNA. As a bivalent DNA vaccine, the VR1012-NH36 was immunoprotective against visceral and cutaneous murine leishmaniasis. In this work we tested the immunotherapy against Leishmania (L.) chagasi infection, using two doses of 100 or 20 microg VR1012-NH36 vaccine (i.m. route), and, as a possible immunomodulator, aqueous garlic extract (8 mg/kg/day by the i.p. route), which was effective in immunotherapy of cutaneous murine leishmaniasis. Liver parasitic load was significantly reduced following treatment with 100 microg (91%) and 20 microg (77%) of the DNA vaccine, and by 20 microg DNA vaccine and garlic extract (76%) (p=0.023). Survival was 33% for saline controls, 100% for the 100 microg vaccine, and 83 and 67% for the 20 microg vaccine with and without garlic extract addition, respectively. Garlic treatment alone did not reduce parasite load (p>0.05), but increased survival (100%). The NH36-DNA vaccine was highly effective as a new tool for the therapy and control of visceral leishmaniasis, while the mild protective effect of garlic might be related to an unspecific enhancement of IFN-gamma secretion.

Acylated and deacylated saponins of Quillaja saponaria mixture as adjuvants for the FML-vaccine against visceral leishmaniasis.

The adjuvant of the FML-vaccine against murine and canine visceral leishmaniasis, the Riedel de Haen saponin mixture, was fractionated by ion exchange chromatography on DEAE-cellulose to afford one TLC homogeneous Quillaja saponaria Molina QS21 saponin fraction (18.0%), a mixture of two deacylsaponins (19.4%), sucrose (39.9%), sucrose and glucose (19.7%), rutin (0.8%) and quercetin (2.2%), that were identified by comparison of 1H and 13C NMR spectroscopy. The QS21 shows the typical aldehyde group in C-23 (65% equatorial) and a normonoterpene moiety acylated in C-28. The deacylsaponins show the aldehyde group but do not have the normonoterpene moiety. Balb/c mice were vaccinated with 150 microg of FML antigen of Leishmania donovani and 100 microg of each obtained fraction and further challenged by infection with 10(8) amastigotes of Leishmania chagasi. The safety analysis and the effect on humoral and cellular immune responses and in clinical signs showed that the QS21 saponin and the deacylsaponins are the most active adjuvant compounds of the Riedel the Haen saponin mixture. Both induced the highest and non-significantly different increases in DTH, CD4+ T lymphocytes in spleen, IFN-gamma in vitro, body weight gain and the most pronounced reduction of parasite burden in liver (95% for QS21 and 86% for deacylsaponins; p>0.05). While the QS21 showed mild toxicity, significant adjuvant effect on the anti-FML humoral response before and after infection, and decrease in liver relative weight, the deacylsaponins showed no toxicity, less haemolysis and antibody and DTH responses increased mainly after infection, still inducing a stronger Leishmania-specific in vitro splenocyte proliferation. Our results confirm in the Riedel de Haen saponin extract the presence of deacylsaponins normonoterpene-deprivated which are non-toxic and capable of inducing a specific and strong immunoprotective response in vaccination against murine visceral leishmaniasis.

Leishmune vaccine blocks the transmission of canine visceral leishmaniasis: absence of Leishmania parasites in blood, skin and lymph nodes of vaccinated exposed dogs.

Leishmune vaccine is the first licensed vaccine against canine visceral leishmaniasis. It contains the Fucose-Mannose-ligand (FML) antigen of Leishmania donovani. The potential Leishmune vaccine effect on the interruption of the transmission of the disease, was assayed by monitoring, in untreated (n=40) and vaccinated dogs (n=32) of a Brazilian epidemic area: the kala-azar clinical signs, the FML-seropositivity and the Leishmania parasite evidence by immunohistochemistry of skin and PCR for Leishmanial DNA of lymph node and blood samples. On month 11 after vaccination, untreated controls showed: 25% of symptomatic cases, 50% of FML-seropositivity, 56.7% of lymph node PCR, 15.7% of blood PCR and 25% of immunohistochemical positive reactions. The Leishmune-vaccinated dogs showed 100% of seropositivity to FML and a complete absence of clinical signs and of parasites (0%) in skin, lymph node and blood PCR samples (p<0.01). The positivity in FML-ELISA in untreated dogs significantly correlates with the PCR in lymph node samples (p<0.001) and with the increase in number of symptoms (p=0.006) being strong markers of infectiousness. The absence of symptoms and of evidence of Leishmania DNA and parasites in Leishmune-vaccinated animals indicates the non-infectious condition of the Leishmune-vaccinated dogs.