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

Processo e composição farmacêutica imunoquimioterápica para tratamento de leishmaníase canina e humana

DepositadaCompreende uma vacina contendo o antígeno FML (Fucose Mannose-Iigand ou Ligante de Fucose e Manose) e o adjuvante saponina, usada em combinação com quimioterápicos, mostrando propriedade curativa, deixando os cães previamente infectados, na condição de cura estéril da leishmaniose visceral e tegumentar, caracterizada por ausência de parasitas e pela ausência total de DNA de Leishmania, visando impedir a transmissão do parasita causador da Leishmaniose canina visceral do cão para o inseto transmissor e desta forma, para outros cães e humanos. A invenção compreende, também, o uso da citada composição para produzir formulações destinadas a tratar a leishmaniose visceral canina e as leishmanioses viscerais e tegumentares murinas, humanas e caninas, bem como kit compreendendo imunoquimioterápicos para tratar as mesmas patologias

The F1F3 Recombinant Chimera of Leishmania donovani-Nucleoside Hydrolase (NH36) and Its Epitopes Induce Cross-Protection Against Leishmania (V.) braziliensis Infection in Mice

Leishmania (V.) braziliensis is the etiological agent of Cutaneous (CL) and Mucocutaneous leishmaniasis (ML) in the New World. CL can be more benign but ML can be severe and disfiguring. Immunity to these diseases include hypersensitivity, an enhanced inflammatory response with strong IFN-γ and TNF-α secretion. Additionally, the production of IL-10 which down modulates the immune response is reduced. The Nucleoside hydrolase (NH36) of Leishmania (L.) donovani is the main antigen of the Leishmune veterinary vaccine and its F3 domain induces a CD4+ T cell-mediated protection against L. (L.) infantum chagasi infection. Prevention of L. (L.) amazonensis infection requires in contrast an additional CD8+ T cell mediated response induced by the F1 domain. Consequently, the F1F3 recombinant chimera, which contains both domains cloned in tandem, optimized the vaccine efficacy against L. (L.) amazonensis mouse infection. We compared the efficacies of NH36, F1, F3, and the FIF3 chimera against L. (V.) braziliensis mouse infection. The F1F3 chimera increased the NH36 specific IgA and response before and after infection and the IgG and IgG3 levels after challenge. It also induced a 49% stronger intradermal response to leishmanial antigen (IDR) than NH36 that was positively correlated to the levels of IFN-γ and TNF-α, IgG, IgG2a, IgG2b, and IgG3 anti-NH36 antibodies. However, stronger Th1 responses with elevated IFN-γ/IL-10 and TNF-α/IL-10 ratios were promoted by the F3 and F1 vaccines and detected in infected controls while the F1F3 chimera promoted the highest IL-10 secretion, which reduced the pathological Th1 response, and characterized the induction of a mixed and/or T-cell regulatory response. We identified the epitopes responsible for these immune responses. The F3 vaccine induced the earliest immunity and after challenge, the F1F3 chimera promoted the highest CD4+ and CD8+ cytokine-secreting T cell responses, and the predominant frequencies of multifunctional CD4+ and CD8+IL-2+TNF-α+IFN-γ+ T cells. Also as observed against L. (L.) amazonensis infection, the F1F3 chimera showed the strongest reduction of the ear lesions sizes induced by L. (V.) braziliensis. Our results confirm the potential use of the F1F3 chimera in a multi-species cross-protective vaccine against L. (V.) braziliensis

Composição compreendendo frações ou sub-frações de promastigotas ou amastigotas de Leishmania denominadas Fucose Mannose Ligand (fml) e saponina, composição para preparar vacinas bloqueadoras da transmissão de Leishmaniose em humanos e animais compreendendo frações ou sub-frações de promastigotas ou amastigotas de Leishmania (fml) e saponina, uso da composição na preparação de vacinas bloqueadoras para impedir a transmissão de Leishmaniose visceral humana ou animal, uso da composição na preparação de reagentes consistindo na administração de frações ou sub-frações de promastigotas ou amastigotas de Leishmania denominadas Fucose Mannose Ligand (fml) e saponina

28/04/1998: Notificação da homologação da desistência do pedido de patente, apresentada pelo depositante, acarretando o encerramento do processo administrativo.DepositadaA invenção trata de uma composição compreendendo frações ou sub-frações de promastigotas ou amastigotas de Leishmania, denominada "Fucose Mannose Ligand" (FML) e saponina. A invenção compreende, também, o uso da composição para preparar vacina bloqueadora impedindo a transmissão de Leishmaniose em humanos ou animais

Cross-protective immunity to Leishmania amazonensis is mediated by CD4+and CD8+epitopes of Leishmania donovani nucleoside hydrolase terminal domains

The nucleoside hydrolase (NH) of Leishmania donovani (NH36) is a phylogenetic marker of high homology among Leishmania parasites. in mice and dog vaccination, NH36 induces a CD4+ T cell-driven protective response against Leishmania chagasi infection directed against its C-terminal domain (F3). the C-terminal and N-terminal domain vaccines also decreased the footpad lesion caused by Leishmania amazonensis. We studied the basis of the crossed immune response using recombinant generated peptides covering the whole NH36 sequence and saponin for mice prophylaxis against L. amazonensis. the F1 (amino acids 1-103) and F3 peptide (amino acids 199-314) vaccines enhanced the IgG and IgG2a anti-NH36 antibodies to similar levels. the F3 vaccine induced the strongest DTH response, the highest proportions of NH36-specific CD4+ and CD8+ T cells after challenge and the highest expression of IFN-gamma and TNF-alpha. the F1 vaccine, on the other hand, induced a weaker but significant DTH response and a mild enhancement of IFN-gamma and TNF-alpha levels. the in vivo depletion with anti-CD4 or CD8 monoclonal antibodies disclosed that cross-protection against L. amazonensis infection was mediated by a CD4+ T cell response directed against the C-terminal domain (75% of reduction of the size of footpad lesion) followed by a CD8+T cell response against the N-terminal domain of NH36 (57% of reduction of footpad lesions). Both vaccines were capable of inducing long-term cross-immunity. the amino acid sequence of NH36 showed 93% identity to the sequence of the NH A34480 of L amazonensis, which also showed the presence of completely conserved predicted epitopes for CD4+ and CD8+ T cells in F1 domain, and of CD4+ epitopes differing by a single amino acid, in F1 and F3 domains. the identification of the C-terminal and N-terminal domains as the targets of the immune response to NH36 in the model of L. amazonensis infection represents a basis for the rationale development of a bivalent vaccine against leishmaniasis.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ)Univ Fed Rio de Janeiro, Inst Microbiol Paulo Goes, Dept Microbiol Geral, Lab Biol & Bioquim Leishmania, BR-21941902 Rio de Janeiro, RJ, BrazilUniv Fed Rio de Janeiro, Inst Microbiol Paulo Goes, Lab Imunol, BR-21941902 Rio de Janeiro, RJ, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Microbiol Imunol & Parasitol, São Paulo, BrazilUniv Fed Minas Gerais, Inst Ciencias Biol, Dept Biol Geral, Belo Horizonte, MG, BrazilUniv Fed Rio de Janeiro, Fac Med, Programa Pos Grad Clin Med, BR-21941902 Rio de Janeiro, RJ, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Ctr Terapia Celular & Mol, Dept Microbiol Imunol & Parasitol, São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Microbiol Imunol & Parasitol, São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Ctr Terapia Celular & Mol, Dept Microbiol Imunol & Parasitol, São Paulo, BrazilCNPq: 301215-2007-3CNPq: 302039/2010-4CNPq: 559756/2010-0CNPq: 404400/2012-4FAPERJ: 102733/2008FAPERJ: 102957/2011FAPERJ: E-26/102415/2010FAPERJ: E-26/110535/2010Web of Scienc

CD4+ Th1 and Th17 responses and multifunctional CD8 T lymphocytes associated with cure or disease worsening in human visceral leishmaniasis

IntroductionIn VL, a proinflammatory phenotype is typically associated with enhanced phagocytosis and a Th1 mediated immune response resulting in infection control. In contrast, an anti-inflammatory phenotype, associated with a predominant regulatory response, typically enables intracellular multiplication of Leishmania parasites and disease progression.MethodsTo investigate the impact of chemotherapy on Th2 and Th17 immune responses in patients with visceral leishmaniasis (VL), we assessed all combinations of intracellular expression of IFN-γ, IL-10, IL-4 and IL-17 in the CD4+ and CD8+ T cell populations of peripheral blood mononuclear cell (PBMC) samples from patients, after antigenic stimulation with Leishmania lysate, throughout treatment and follow-up. As increases in spleen and liver sizes and decreases in hematocrit, hemogloblin, erythrocytes, monocytes, leukocytes and platelets levels are strongly related to the disease, we studied the correlations between the frequencies of T cells producing the afore mentioned cytokines, individually and in combination, and these variables, as markers of disease or cure.ResultsWe found that the frequency of IFN-γ-producingCD4+ T cells increased until the end of chemotherapy with Glucantime® or AmBisome ®, while IL-10, IL-4 and IL-17-producing CD4+ T cells peaked on day 7 following the start of treatment. Although the frequency of CD4+IL-17+ cells decreased during treatment an increase was observed after clinical cure. The frequency of CD4+ T cells producing only IFN-γ or IL-17 correlated with blood monocytes levels. Frequencies of double-producers of IFN-γ and IL-10 or IL-4 correlated positively with eosinophils and platelets levels. Together, this suggest that IFN-γ drives the immune response towards Th1 at cure. In contrast, and associated with disease or Th2 response, the frequency of CD4+ IL-10+ cells correlated positively with spleen sizes and negatively with circulating monocyte levels, while the frequency of CD4+ producing both IL-4 and IL-10 correlated negatively with platelets levels. The frequency of CD8+ single-producers of IFN-γ increased from day 21 to 90 while that of single-producers of IL-10 peaked on day 7, of IL-4 on day 30 and of IL-17, on day 180. IFN-γ expression in CD8+ single- and double-producers of cytokines was indicative of an immune response associated with cure. In contrast, frequencies of CD8+ double-producers of IL-4 and IL-10, IL-4 and IL-17 and IL-10 and IL-17 and producers of three and four cytokines, were associated with disease and were low after the cure. Frequencies of CD8+ T cells producing IFN-γ alone or with IL-17 were positively correlated with platelets levels. In contrast, as markers of disease: 1) frequencies of single producers of IL-10 correlated negatively with leukocytes levels, 2) frequencies of double producers of IL-4 and IL-10 correlated negatively with platelet, leukocyte, lymphocyte and circulating monocyte levels, 3) frequencies of triple-producers of IFN-γ, IL-4 and IL-10 correlated negatively with platelet, leukocyte and neutrophil levels and 4) frequencies of producers of IFN-γ, IL-4, IL-10 and IL-17 simultaneously correlated positively with spleen size, and negatively with leukocyte and neutrophil levels.DiscussionOur results confirmed that the clinical improvement of VL patients correlates with the decrease of an IL-4 and IL-10 CD4+Th2 response, the recovery of CD4+ Th1 and Th17 responses and the frequency of CD8+ single-producers of IFN-γ and double producers of IFN-γ and IL-17

Adaptive Immunity against Leishmania Nucleoside Hydrolase Maps Its C-Terminal Domain as the Target of the CD4+ T Cell–Driven Protective Response

Nucleoside hydrolases (NHs) show homology among parasite protozoa, fungi and bacteria. They are vital protagonists in the establishment of early infection and, therefore, are excellent candidates for the pathogen recognition by adaptive immune responses. Immune protection against NHs would prevent disease at the early infection of several pathogens. We have identified the domain of the NH of L. donovani (NH36) responsible for its immunogenicity and protective efficacy against murine visceral leishmaniasis (VL). Using recombinant generated peptides covering the whole NH36 sequence and saponin we demonstrate that protection against L. chagasi is related to its C-terminal domain (amino-acids 199–314) and is mediated mainly by a CD4+ T cell driven response with a lower contribution of CD8+ T cells. Immunization with this peptide exceeds in 36.73±12.33% the protective response induced by the cognate NH36 protein. Increases in IgM, IgG2a, IgG1 and IgG2b antibodies, CD4+ T cell proportions, IFN-γ secretion, ratios of IFN-γ/IL-10 producing CD4+ and CD8+ T cells and percents of antibody binding inhibition by synthetic predicted epitopes were detected in F3 vaccinated mice. The increases in DTH and in ratios of TNFα/IL-10 CD4+ producing cells were however the strong correlates of protection which was confirmed by in vivo depletion with monoclonal antibodies, algorithm predicted CD4 and CD8 epitopes and a pronounced decrease in parasite load (90.5–88.23%; p = 0.011) that was long-lasting. No decrease in parasite load was detected after vaccination with the N-domain of NH36, in spite of the induction of IFN-γ/IL-10 expression by CD4+ T cells after challenge. Both peptides reduced the size of footpad lesions, but only the C-domain reduced the parasite load of mice challenged with L. amazonensis. The identification of the target of the immune response to NH36 represents a basis for the rationale development of a bivalent vaccine against leishmaniasis and for multivalent vaccines against NHs-dependent pathogens

One Health: The global challenge of epidemic and endemic leishmaniasis

'One Health' proposes the unification of medical and veterinary sciences with the establishment of collaborative ventures in clinical care, surveillance and control of cross-species disease, education, and research into disease pathogenesis, diagnosis, therapy and vaccination. The concept encompasses the human population, domestic animals and wildlife, and the impact that environmental changes ('environmental health') such as global warming will have on these populations. Visceral leishmaniasis is a perfect example of a small companion animal disease for which prevention and control might abolish or decrease the suffering of canine and human patients, and which aligns well with the One Health approach. In this review we discuss how surveillance for leishmaniases is undertaken globally through the control of anthroponootic visceral leishmaniasis (AVL) and zoonotic visceral leishmaniasis (ZVL). The ZVL epidemic has been managed to date by the culling of infected dogs, treatment of human cases and control of the sandfly vector by insecticidal treatment of human homes and the canine reservoir. Recently, preventive vaccination of dogs in Brazil has led to reduction in the incidence of the canine and human disease. Vaccination permits greater dog owner compliance with control measures than a culling programme. Another advance in disease control in Africa is provided by a surveillance programme that combines remote satellite sensing, ecological modelling, vector surveillance and geo-spatial mapping of the distribution of vectors and of the animal-to-animal or animal-to-human pathogen transmission. This coordinated programme generates advisory notices and alerts on emerging infectious disease outbreaks that may impede or avoid the spreading of visceral leishmaniasis to new areas of the planet as a consequence of global warming