Edilma Paraguai de Souza

A phase III trial of efficacy of the FML-vaccine against canine kala-azar in an endemic area of Brazil (São Gonçalo do Amaranto, RN)

Protection against canine kala-azar was investigated in naturally exposed dogs of an endemic area, vaccinated with the fucose mannose ligand (FML)-vaccine of Leishmania donovani. A total of 97% of vaccinees were seropositive to FML and 100% showed intradermal reaction to L. donovani lysate, 7 months after vaccination. The absorbency values and size of intradermal reaction were both significantly higher in vaccinees than in controls (ANOVA, P<0.0001). After 2 years, 92% (chi(2)=6.996; P<0.0025) protection was achieved: only 8% of vaccinees showed mild signs of kala-azar with no deaths while 33% of controls developed clinical or fatal disease. The FML-vaccine induced a significant, long-lasting and strong protective effect against canine kala-azar in the field.

Effective immunotherapy against canine visceral leishmaniasis with the FML-vaccine

Abstract The potential effect of the fucose mannose ligand (FML)-vaccine on immunotherapy of canine visceral leishmaniasis was assayed on five mongrel dogs experimentally infected with Leishmania donovani and on 21 Leishmania chagasi naturally infected dogs when seropositive to FML but completely asymptomatic. The clinical signs of the experimentally infected, symptomatic dogs only disappeared after the complete vaccination. Protection was obtained in 3/5 animals that remained asymptomatic, IDR positive and parasite free, 1 year after infection. Furthermore, the asymptomatic, FML-vaccine treated dogs showed stable anti-FML IgG1 levels, increasing IgG2 levels and 79–95% of positive DTH response, during the whole experiment. Twenty-two months after complete vaccination, no obits due to visceral leishmaniasis were recorded and 90% of these dogs were still asymptomatic, healthy and parasite free. On the other hand, 37% (17/46 dogs) kala-azar obits were recorded in a control group that received no treatment during the same period, and that was FML-seropositive and asymtpomatic at the beginning of the assay. Our results indicate that the FML-vaccine was effective in the immunotherapy against visceral leishmaniasis of asymptomatic infected dogs. Normal proportions of CD4 and CD21 lymphocytes were detected in PBMC by FACS analysis, in dogs submitted to immunotherapy, suggesting their non-infectious condition. All animals showed as well significantly increased percents of CD8 lymphocytes as expected for Quillaja saponin (QuilA) vaccine treatments.

Cross-Protective Efficacy of a Prophylactic Leishmania donovani DNA Vaccine against Visceral and Cutaneous Murine Leishmaniasis

ABSTRACT The fucose-mannose ligand (FML) complex of Leishmania donovani is a promising vaccine candidate against murine and canine visceral leishmaniasis, and its main component is a 36-kDa nucleoside hydrolase (NH36). In this study, we tested the immune response and protection induced by the purified FML, the recombinant NH36 (rNH36), and NH36 DNA vaccines against the agents of visceral (L. chagasi) and cutaneous (L. mexicana) leishmaniasis in BALB/c mice. Mice developed weak humoral response to the vaccines alone, except for those immunized with FML. However, all three vaccine groups presented elevated immunoglobulin G (IgG), IgG1, and IgG2a levels after infection with L. chagasi, whereas no differences were observed between vaccine and control groups after infection with L. mexicana. A strong intradermal reaction to L. donovani and L. mexicana antigens was observed in mice immunized with rNH36 or FML, whereas mice immunized with NH36 DNA only reacted against L. donovani antigens. Experimental infection of immunized mice demonstrated that FML and rNH36 induced significant protection against L. chagasi infection with reductions in parasite loads of 79%. FML also conferred partial protection against L. mexicana infection. The best protection was observed in mice immunized with the VR1012-NH36 DNA vaccine, which induced an 88% reduction in L. chagasi parasite load and a 65% reduction in L. mexicana lesion size. Fluorescence-activated cell sorting analysis indicated the DNA vaccine induced a two- to fivefold increase in gamma interferon-producing CD4+ T cells, indicating a Th1-type immune response. Our results showed that the NH36 DNA vaccine induced a strong immunoprotection against visceral and cutaneous leishmaniasis, suggesting that this DNA vaccine represents a very good candidate for use against several Leishmania species.

Saponins, IL12 and BCG adjuvant in the FML-vaccine formulation against murine visceral leishmaniasis

The FML antigen of Leishmania donovani, in combination with either Riedel de Haën (R), QuilA, QS21 saponins, IL12 or BCG, was used in vaccination of an outbred murine model against visceral leishmaniasis (VL). Significant and specific increases in anti-FML IgG and IgM responses were detected for all adjuvants, and in anti-FML IgG1, IgG2a and IgG2b and delayed type of hypersensitivity to L. donovani lysate (DTH), only for all saponins and IL12. The QS21-FML and QuilA-FML groups achieved the highest IgG2a response. QuilA-FML developed the strongest DTH and QS21-FML animals showed the highest serum IFN-gamma concentrations. The reduction of parasitic load in the liver in response to each FML-vaccine formulation was: 52% (P<0.025) for BCG-FML, 73% (P<0.005) for R-FML, 93% (P<0.005) for QuilA-FML and 79.2% (P<0.025) for QS21-FML treated animals, respectively. Protection was specific for R-FML and QS21-FML while the QuilA saponin treatment itself induced 69% of LDU reduction. The FML-saponin vaccines promote significant, specific and strong protective effects against murine visceral leishmaniasis. BCG-FML induced minor and non-specific protection while IL12-FML, although enhancing the specific antibody and IDR response, failed to reduce the parasitic load of infected animals.

IgG1/IgG2 antibody dichotomy in sera of vaccinated or naturally infected dogs with visceral leishmaniosis

Canine antibody IgG, IgG1 and IgG2 anti-FML responses were investigated in dogs vaccinated with the fucose-mannose ligand (FML)-vaccine of Leishmania donovani and in dogs with naturally acquired visceral leishmaniosis. While similar levels of total IgG antibodies were seen in the seropositive naturally infected dogs and in vaccinees, significant differences between the groups were found regarding their IgG1/IgG2 anti-FML antibody composition (P<0.005). Higher IgG1 absorbencies were seen in infected dogs, while the IgG2 subtype was predominant in pre-immune sera, and in vaccinated animals, both after the first and the third dose (P<0.005). The average ratio between IgG1/IgG2 was then 1.124 for infected animals and 0.733 for FML-vaccinees. Also, a significant increase in IgG2 antibodies was observed from the first to the third vaccine injection (P<0.005). In the infected dogs, a high correlation between their IgG absorbance (Abs) values and the number of symptoms (P=0.017) was disclosed. Thus, the analysis of IgG subclasses disclosed a dichotomous response to visceral leishmaniosis: IgG1 associated to natural infection and IgG2 associated to a humoral response subsequent to the FML-vaccine treatment. An IgG1/IgG2>or=1 would characterize the sera of visceral leishmaniasis infected animals evoluting towards the overt disease while ratios <or=1 would characterize the sera response of vaccinated protected dogs.

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 Freunds 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.

Immunotherapy against murine experimental visceral leishmaniasis with the FML-vaccine

The fucose mannose ligand (Leishmania donovani FML)-saponin vaccine has earlier shown its immunoprophylactic potential against visceral leishmaniasis in the CB hamster (87.7% of parasite load reduction), Balb/c (84.4%) and Swiss albino mouse (85-93%) models. In this investigation its specific immunotherapeutic efficacy against L. donovani infection in Balb/c mice was studied. The effects of vaccine treatment on the humoral response, delayed type of hypersensitivity to promastigote lysate (DTH), cytokine levels in sera and reduction of the liver parasitic load of L. donovani infected mice, were examined. The types and subtypes of anti-FML antibodies increased significantly in the vaccinees over the saline and saponin controls. As expected for a saponin vaccine, the highest ratios were found in relation to IgG1, IgG2a and IgG2b (4.4, 5 and 2.5, respectively). The DTH response and the in vitro ganglion cell proliferative response against FML antigen were also significantly higher than controls (P<0.005). Concomitantly, an impressive and specific decrease of liver parasitic burden was detected only in vaccine-treated animals (94.7%). Our results indicate that the therapeutic FML-vaccine has a potent effect on modulation of the murine infection leading to the reduction of parasitic load and signs of disease, being a new potential tool in the therapy and control of visceral leishmaniasis.

Nucleoside hydrolase from Leishmania (L.) donovani is an antigen diagnostic for visceral leishmaniasis

Debora M. Santana a,b, Gulnara P. Borja-Cabrera b, Edilma Paraguai de Souza b, Nancy R. Sturm a, Clarisa B. Palatnik de Sousa b, David A. Campbell * a Department of Microbiology, Immunology and Molecular Genetics, School of Medicine, University of California, 10833 Le Conte Avenue, Los Angeles, CA 90095-1747, USA b Instituto de Microbiologia*/Professor Paulo de Goes, CCS, Cidade Universitaria, Ilha do Fundao, UFRJ, CP 68040, CEP 21944-590 Rio de Janeiro, RJ, Brazil

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.