Clarisa B. Palatnik-de-Sousa

Vaccines for leishmaniasis in the fore coming 25 years.

Human vaccination against leishmaniasis using live Leishmania was used in Middle East and Russia (1941-1980). First-generation vaccines, composed by killed parasites induce low efficacies (54%) and were tested in humans and dogs Phase III trials in Asia and South America since 1940. Second-generation vaccines using live genetically modified parasites, or bacteria or viruses containing Leishmania genes, recombinant or native fractions are known since the 1990s. Due to the loss of PAMPs, the use of adjuvants increased vaccine efficacies of the purified antigens to 82%, in Phase III dog trials. Recombinant second-generation vaccines and third-generation DNA vaccines showed average values of parasite load reduction of 68% and 59% in laboratory animal models, respectively, but their success in field trials had not yet been reported. This review is focused on vaccine candidates that show any efficacy against leishmaniasis and that are already in different phase trials. A lot of interest though was generated in recent years, by the studies going on in experimental models. The promising candidates may find a place in the forth coming years. Among them most probably are the multiple-gene DNA vaccines that are stable and do not require cold-chain transportation. In the mean time, second-generation vaccines with native antigens and effective adjuvants are likely to be licensed and used in Public Health control programs in the fore coming 25 years. To date, only three vaccines have been licensed for use: one live vaccine for humans in Uzbekistan, one killed vaccine for human immunotherapy in Brazil and a second-generation vaccine for dog prophylaxis in Brazil.

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.

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.

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.

Vaccines for Canine Leishmaniasis

Leishmaniasis is the third most important vector-borne disease worldwide. Visceral leishmaniasis (VL) is a severe and frequently lethal protozoan disease of increasing incidence and severity due to infected human and dog migration, new geographical distribution of the insect due to global warming, coinfection with immunosuppressive diseases, and poverty. The disease is an anthroponosis in India and Central Africa and a canid zoonosis (ZVL) in the Americas, the Middle East, Central Asia, China, and the Mediterranean. The ZVL epidemic has been controlled by one or more measures including the culling of infected dogs, treatment of human cases, and insecticidal treatment of homes and dogs. However, the use of vaccines is considered the most cost–effective control tool for human and canine disease. Since the severity of the disease is related to the generation of T-cell immunosuppression, effective vaccines should be capable of sustaining or enhancing the T-cell immunity. In this review we summarize the clinical and parasitological characteristics of ZVL with special focus on the cellular and humoral canine immune response and review state-of-the-art vaccine development against human and canine VL. Experimental vaccination against leishmaniasis has evolved from the practice of leishmanization with living parasites to vaccination with crude lysates, native parasite extracts to recombinant and DNA vaccination. Although more than 30 defined vaccines have been studied in laboratory models no human formulation has been licensed so far; however three second-generation canine vaccines have already been registered. As expected for a zoonotic disease, the recent preventive vaccination of dogs in Brazil has led to a reduction in the incidence of canine and human disease. The recent identification of several Leishmania proteins with T-cell epitopes anticipates development of a multiprotein vaccine that will be capable of protecting both humans and dogs against VL.

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

Abstract 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.

Improving methods for epidemiological control of canine visceral leishmaniasis based on a mathematical model. Impact on the incidence of the canine and human disease

The mathematical model described by Dye (1996) condemned the epidemiological canine visceral leishmaniasis control campaign, considering it non-efficient. Using this model, we mathematically demonstrate that the control is not efficient, only at low kappa values (rate at which latent and infectious dogs are lost by the destruction program) which match the canine seropositivity observed in the field by the immunofluorescency (IF) blood eluates analysis. With higher k values, corresponding to IF (kappa = 0.07) or ELISA (kappa = 0.25) results in sera samples, the number of infectious dogs declines to a Ro =1 or Ro =0, respectively, interrupting the transmission and the advancement of epidemics. We also experimentally demonstrate that the dog removal, following the results of IF of sera, instead of eluates lead to a 57% (p < 0.005) decrease in canine cases and 87.5% (p < 0.005) in human cases. Our mathematical and experimental results indicate that the control campaign become more efficient by enhancing the sensitivity of the diagnostic assay.

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.

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.