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.

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.

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.

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.

Vaccination of Balb/c mice against experimental visceral leishmaniasis with the GP36 glycoprotein antigen of Leishmania donovani.

Leishmania donovani GP36 glycoprotein is the main antigen of the FML Fucose Mannose Ligand (FML) complex specifically recognized by sera of kala-azar human patients. The GP36 was isolated by chemical elution + sonication and used for Balb/c mouse vaccination in combination with saponin, by the s.c. route, inducing a strong and specific protective effect against experimental visceral leishmaniasis shown by the increase of: specific IgG antibodies (82.6%), mainly IgG2a, the delayed type of hypersensitivity to promastigote lysate (37.8%, P < 0.001), the in vitro cellular proliferative response to GP36 of ganglia lymphocytes (53.5%, P < 0.005) and the decrease of liver parasite burden (68.1%, P < 0.025). Saponin treated controls reacted significantly differently from GP36 vaccinated animals at all the assayed variables (P < 0.05). GP36 induced significant protection against murine visceral leishmaniasis at concentrations commonly used for vaccination with recombinant antigens.

Vaccination of Swiss Albino mice against experimental visceral leishmaniasis with the FML antigen of Leishmania donovani.

The FML antigen of Leishmania donovani in combination with saponin, aluminum hydroxide (Al(OH)3) and Freund's incomplete adjuvant (FIA) was used in vaccines tested in an outbred murine model of visceral leishmaniasis, either through intraperitoneal or subcutaneous routes. The humoral response was significantly higher in the groups treated with FML + saponin or FML + Al(OH)3 than in controls, both before and after the infection. Animals immunized by the i.p. route developed higher antibody titres. A significant and specific reduction of parasitic load in relation to saline (85%, p < 0.01) and saponin (p < 0.025) controls, was seen in animals treated with FML + saponin by the i.p. Coincidentally with this reduction, an increase in antibodies of the IgG2a subtype was detected only in animals treated with FML + saponin i.p. A reduction of 88% in parasitic load was achieved by the combination of FML + Al(OH)3 (s.c.), but the Al(OH)3 treatment itself accounted for 68% of this protection. In our conditions, vaccination with FML + saponin i.p. was superior to other treatments and had no toxic effect due to saponin.

Long lasting protection against canine kala-azar using the FML-QuilA saponin vaccine in an endemic area of Brazil (São Gonçalo do Amarante, RN).

Naturally exposed dogs of an endemic area were vaccinated with the fucose mannose ligand (FML) antigen of Leishmania donovani in formulation with QuilA saponin. The 100% of vaccinees were seropositive to FML and showed intradermal reaction to L. donovani lysate, 2 months after vaccination. The absorbency values and size of intradermal reaction were both significantly higher in vaccinees than in controls along a 3.5 years period (ANOVA, P<0.0001). The 25% of the control animals (two dogs on the first year and six dogs on the fourth year, respectively) and 5% of the vaccinees (one dog during the fourth year) developed clinical and fatal disease until the end of experiment. This difference was significant (chi(2)=3.93, P<0.05). This means that 95% protection against kala-azar was achieved in vaccinees, after FML-QuilA vaccination (80% of vaccine efficacy (VE)). Leishmania infection was also confirmed, 3.5 years after vaccination, in saline controls that showed positive polymerase chain reaction (PCR) for Leishmania DNA and FML-serology with no intradermal reaction. Higher seropositivities and intradermal reactions with no Leishmanial DNA were detected in vaccinees. The FML-QuilA vaccine induced a significant, long lasting and strong protective effect against canine kala-azar in the field.

Protective vaccination against murine visceral leishmaniasis using aldehyde-containing Quillaja saponaria sapogenins.

The presence of aldehyde groups at C-23 and C-24 of the triterpen aglycon moiety was disclosed in 1H NMR spectra of both the Riedel de Haen saponin (R) (delta 9.336) and Quillaja saponaria QuilA saponin (delta 9.348). The sign of the C-28 acylated linked moiety (delta 176) was present in both saponins, while the delta 171 at C-28 (carboxy group) corresponding to the deacylated saponin, was only detected in the QuilA preparation, indicating 50% of hydrolysis of the ester moiety, probably due to the storage in aqueous solution. The normoterpen moiety was present in both saponins (signals at delta 14-18). The chemical removal of saponin glicidic moieties gave rise to their sapogenin fractions. Their 1H NMR spectra showed the presence of two signals (delta 9.226 and 9.236) for sapogenin R and two signals (delta 9.338 and 9.352) for the QuilA sapogenin. The intensity of the signals suggested two conformational isomers of sapogenin R in the ratio 53% of equatorial aldehyde group to 47% of axial aldehyde group, and two conformational isomers of QuilA sapogenin in the ratio 76% of equatorial aldehyde group to 24% of axial aldehyde group. The chemical treatment abolished the saponin slight in vivo toxicity, reduced their hemolytic potential, did not affect their aldehyde contents, but gave rise to an enriched axial aldehyde-containing sapogenin R with enhanced potential on antibody humoral response (anti-IgM, IgG, IgG1, IgG2a, IgG2b and IgG3) and to an enriched equatorial aldehyde-containing QuilA-sapogenin that induced a mainly cellular specific immune response (increased intradermal response to leishmanial antigen and IFNgamma sera levels) and effective protection against murine infection by L. donovani (77% reduction in liver parasitic load). Our results suggest that the Riedel de Haen saponin is probably a Quillaja saponaria saponin.

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 transfused 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 in endemic.