Marcos Palatnik

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.

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.

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.

Haemolytic activities of plant saponins and adjuvants. Effect of Periandra mediterranea saponin on the humoral response to the FML antigen of Leishmania donovani

An 87.7% (P < 0.01) and 84% (P < 0.001) of protection against visceral leishmaniasis was achieved in CB hamsters and Balb/c mice, respectively, with saponin combined to the fucose-mannose ligand of Leishmania donovani (FML). However, an undesirable haemolytic effect was described for several saponins. Aiming to improve the formulation with FML/saponin, we comparatively analysed the haemolytic potential of recently characterized plant saponins and currently used adjuvants. The haemolytic activity of steroidic saponins from Agave sisalana; Smilax officinalis as well as commercial saponin (Riedel De Haëns), was higher than that of triterpenoid ones (Bredemeyera floribunda; Periandra mediterranea) and the Freunds complete adjuvant. The concentration resulting in 50% haemolysis was 500 micrograms ml-1 for aluminum hydroxide. The low haemolytic effect of P. mediterranea saponin was abolished by removal of its glycidic moiety and its sapogenin fraction as well as the Freunds Incomplete Adjuvant were non-haemolytic within this range. Furthermore, the adjuvant effect of three doses of P. mediterranea saponin injected with the FML antigen of L. donovani, was assayed in mice, either by the intraperitoneal (i.p.) or the subcutaneous (s.c.) route. The anti-FML IgG antibody levels increased and detectable levels were observed up to 3 months in the s.c. group. The response was expanded in both groups after an injection with a fourth vaccine dose. The IgG response showed increased levels of IgG2a only in the i.p. group, while IgG2b and IgG1 but not IgG3 antibodies were higher than controls in both groups. In conclusion, the results suggest that the recently described triterpenoid fractions of P. mediterranea can be safely used as adjuvant with low or non-haemolytic effect.

Decrease of the incidence of human and canine visceral leishmaniasis after dog vaccination with Leishmune® in Brazilian endemic areas.

Leishmune, the first prophylactic vaccine licensed against canine visceral leishmaniasis (CVL), has been used in Brazil since 2004, where seropositive dogs are sacrificed in order to control human visceral leishmaniasis (VL). We demonstrate here that vaccination with Leishmune does not interfere with the serological control campaign (110,000 dogs). Only 1.3% of positivity (76 among 5860) was detected among Leishmune uninfected vaccinees. We also analyzed the possible additive effect of Leishmune vaccination over dog culling, on the decrease of the incidence of CVL and VL in two Brazilian endemic areas, from 2004 to 2006. In Araçatuba, a 25% of decline was seen in CVL with a 61% decline in human cases, indicating the additive effect of Leishmune vaccination of 5.7% of the healthy dogs (1419 dogs), on regular dog culling. In Belo Horizonte (BH), rising curves of canine and human incidence were observed in the districts of Barreiro, Venda Nova and Noroeste, while the canine and human incidence of Centro Sul, Leste, Nordeste, Norte, Pampulha and Oeste, started to decrease or maintained a stabilized plateau after Leishmune vaccination. Among the districts showing a percent decrease of human incidence (-36.5%), Centro Sul and Pampulha showed the highest dog vaccination percents (63.27% and 27.27%, respectively) and the lowest dog incidence (-3.36% and 1.89%, respectively). They were followed by Oeste, that vaccinated 25.30% of the animals and experienced an increase of only 12.86% of dog incidence and by Leste and Nordeste, with lower proportions of vaccinees (11.72% and 10.76%, respectively) and probably because of that, slightly higher canine incidences (42.77% and 35.73%). The only exception was found in Norte district where the reduced human and canine incidence were not correlated to Leishmune vaccination. Much lower proportions of dogs were vaccinated in Venda Nova (4.35%), Noroeste (10.27%) and Barreiro (0.09%) districts, which according to that exhibited very increased canine incidences (24.48%, 21.85% and 328.57%, respectively), and pronounced increases in human incidence (14%, 4% and 17%, respectively). The decrease of canine (p=-0.008) and human incidences (p=-0.048) is directly correlated to the increase of the number of vaccinated dogs, confirming the additive control effect of Leishmune vaccination over dog culling, reducing the parasite reservoir, protecting dogs and, in this way, reducing the risk of transmission of VL to humans and becoming a new effective control tool.

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.

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

Abstract 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 dogs potential infectiosity to phlebotomines. The enriched-Leishmune® vaccine was subjected to a safety analysis and found to be well tolerated and safe.

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.

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.