Treatment of visceral leishmaniasis

I am deeply aware of the honor you have conferred upon me by election to the presidency of the Western Surgical Association and I would like to take this opportunity to express my deepest appreciation. I would like to add my grateful thanks, also, to the various officers of this association who so ably conduct the affairs of the society, and to the members of the Local Committee who have carried the burden of arrangements for this meeting, and to all of you for your continuous cooperation and interest which make the meetings of this society so stimulating. Although the major surgical associations of today are relatively new in the history of scientific societies, most of them in this country have, so far, withstood the test of time and are functioning today with vigor and usefulness. The broad objectives and practical aims of these flourishing organizations have been important factors

Progress and Problems

Diffuse cutaneous leishmaniasis (DCL) is a rare clinical manifestation of tegumentary leishmaniasis. The molecular mechanisms underlying DCL pathogenesis remain unclear, and there is no efficient treatment available. This study investigated the systemic and in situ expression of the inflammatory response that might contribute to suppression in DCL. The plasma levels of arginase I, ornithine decarboxylase (ODC), transforming growth factor β (TGF-β), and prostaglandin E2 (PGE2) were higher in patients with DCL, compared with patients with localized cutaneous leishmaniasis (LCL) or with controls from an area of endemicity. In situ transcriptomic analyses reinforced the association between arginase I expression and enzymes involved in prostaglandin and polyamine synthesis. Immunohistochemistry confirmed that arginase I, ODC, and cyclooxygenase2 expression was higher in lesion biopsy specimens from patients with DCL than in those from patients with LCL. Inhibition of arginase I or ODC abrogates L. amazonensis replication in infected human macrophages. Our data implicate arginase I, ODC, PGE2, and TGF-β in the failure to mount an efficient immune response and suggest perspectives in the development of new strategies for therapeutic intervention for patients with DCL.

Arginase I, polyamine, and prostaglandin E2 pathways suppress the inflammatory response and contribute to diffuse cutaneous leishmaniasis (vol 211, pg 426, 2015)

Visceral leishmaniasis (VL), caused by Leishmania (L.) donovani and L. infantum protozoan parasites, can provoke overwhelming and protracted epidemics, with high case–fatality rates. An effective vaccine against the disease must rely on the generation of a strong and long-lasting T cell immunity, mediated by CD4+ TH1 and CD8+ T cells. Multi-epitope peptide-based vaccine development is manifesting as the new era of vaccination strategies against Leishmania infection. In the present study, we designed chimeric peptides containing HLA-restricted epitopes from three immunogenic L. infantum proteins (cysteine peptidase A, histone H1 and kinetoplastid membrane protein 11), in order to be encapsulated in poly(lactic-co-glycolic) acid nanoparticles with or without the adjuvant Monophosphoryl lipid A (MPLA) or surface modification with an octapeptide targeting the Tumor Necrosis Factor Receptor II. We aimed to construct differentially functionalized peptide-based nanovaccine candidates and investigate their capacity to stimulate the immunomodulatory properties of dendritic cells (DCs), which are critical regulators of adaptive immunity generated upon vaccination. According to our results, DCs stimulation with the peptide-based nanovaccine candidates with MPLA incorporation or surface modification induced an enhanced maturation profile with prominent IL-12 production, promoting allogeneic Τ cell proliferation and intracellular production of IFN by CD4+ and CD8+ T cell subsets. In addition, DCs stimulated with the peptide-based nanovaccine candidate with MPLA incorporation exhibited a robust transcriptional activation, characterized by up-regulated genes indicative of vaccine-driven DCs differentiation towards type 1 phenotype. Immunization of HLA A2.1 transgenic mice with this peptide-based nanovaccine candidate induced peptide-specific IFNγ-producing CD8+ T cells and conferred significant protection against L. infantum infection. Concluding, our findings supported that encapsulation of more than one chimeric multi-epitope peptides from different immunogenic L. infantum proteins in a proper biocompatible delivery system with the right adjuvant is considered as an improved promising approach for the development of a vaccine against VL.

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A Poly(Lactic-co-Glycolic) Acid Nanovaccine Based on Chimeric Peptides from Different Leishmania infantum Proteins Induces Dendritic Cells Maturation and Promotes Peptide-Specific IFNγ-Producing CD8+ T Cells Essential for the Protection against Experimental Visceral Leishmaniasis.

Transition state theory teaches that chemically stable mimics of enzymatic transition states will bind tightly to their cognate enzymes. Kinetic isotope effects combined with computational quantum chemistry provides enzymatic transition state information with sufficient fidelity to design transition state analogues. Examples are selected from various stages of drug development to demonstrate the application of transition state theory, inhibitor design, physicochemical characterization of transition state analogues, and their progress in drug development.

Enzymatic Transition States and Drug Design

The Leishmania donovani-nucleoside hydrolase (NH36) and NH A34480 of L. amazonensis share 93% of sequence identity. In mice, the NH36 induced protection against visceral leishmaniasis is mediated by a CD4+ T cell response against its C-terminal domain (F3). Besides this CD4+ Th1 response, prevention and cure of L. amazonensis infection require also additional CD8+ and regulatory T-cell responses to the NH36 N-terminal (F1 domain). We investigated if mice vaccination with F1 and F3 domains cloned in tandem, in a recombinant chimera, with saponin, optimizes the vaccine efficacy against L. amazonensis infection above the levels promoted by the two admixed domains or by each domain independently. The chimera induced the highest IgA, IgG and IgG2a anti-NH36 antibody, IDR, IFN-γ and IL-10 responses, while TNF-α was more secreted by mice vaccinated with F3 or all F3-contaning vaccines. Additionally, the chimera and the F1 vaccine also induced the highest proportions of CD4+ and CD8+ T cells secreting IL-2, TNF-α or IFN-γ alone, TNF-α in combination with IL-2 or IFN-γ and of CD4+ multifunctional cells secreting IL-2, TNF-α and IFN-γ. Correlating with the immunological results the strongest reductions of skin lesions sizes were determined by the admixed domains (80 %) and by the chimera (84 %), which also promoted the most pronounced and significant reduction of the parasite load (99.8 %). Thus, the epitope presentation in a recombinant chimera optimizes immunogenicity and efficacy above the levels induced by the independent or admixed F1 and F3 domains. The multiparameter analysis disclosed that the Th1-CD4+ T helper response induced by the chimera is mainly directed against its FRYPRPKHCHTQVA epitope. Additionally, the YPPEFKTKL epitope of F1 induced the second most important CD4+ T cell response, and, followed by the DVAGIVGVPVAAGCT, FMLQILDFYTKVYE and ELLAITTVVGNQ sequences, also the most potent CD8+ T cell responses and IL-10 secretion. Remarkably, the YPPEFKTKL epitope shows high amino acid identity with a multipotent PADRE sequence and stimulates simultaneously the CD4+, CD8+ T cell and a probable T regulatory response. With this approach we advanced in the design of a nucleoside hydrolase NH36 polytope vaccine capable of inducing cross-protection to cutaneous leishmaniasis.

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A Chimera Containing CD4+ and CD8+ T-Cell Epitopes of the Leishmania donovani Nucleoside Hydrolase (NH36) Optimizes Cross-Protection against Leishmania amazonesis Infection

The Nucleoside hydrolase 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-γ and TNF-α. The F1 vaccine, on the other hand, induced a weaker but significant DTH response and a mild enhancement of IFN-γ and TNF-α 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 in 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. amazonesis infection represents a basis for the rationale development of a bivalent vaccine against leishmaniasis.

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Cross-Protective Immunity to Leishmania amazonensis is Mediated by CD4+ and CD8+ Epitopes of Leishmania donovani Nucleoside Hydrolase Terminal Domains.

Background 
Immucillins ImmA (IA), ImmH (IH) and SerMe-ImmH (SMIH) are synthetic deazapurine nucleoside analogues that inhibit Leishmania (L.) infantum chagasi and Leishmania (L.) amazonensis multiplication in vitro without macrophage toxicity. Immucillins are compared to the Glucantime standard drug in the chemotherapy of Leishmania (L.) infantum chagasi infection in mice and hamsters. These agents are tested for toxicity and immune system response.


Methodology/Principal Findings 
BALB/c mice were infected with 107 amastigotes, treated with IA, IH, SMIH or Glucantime (2.5mg/kg/day) and monitored for clinical variables, parasite load, antibody levels and splenocyte IFN-γ, TNF-α, and IL-10 expression. Cytokines and CD4+, CD8+ and CD19+ lymphocyte frequencies were assessed in uninfected controls and in response to immucillins. Urea, creatinine, GOT and GPT levels were monitored in sera. Anti-Leishmania-specific IgG1 antibodies (anti-NH36) increased in untreated animals. IgG2a response, high levels of IFN-γ, TNF-α and lower levels of IL-10 were detected in mice treated with the immucillins and Glucantime. Immucillins permitted normal weight gain, prevented hepato-splenomegaly and cleared the parasite infection (85–89%) without renal and hepatic toxicity. Immucillins promoted 35% lower secretion of IFN-γ in uninfected controls than in infected mice. IA and IH increased the CD4+ T and CD19+ B cell frequencies. SMIH increased only the proportion of CD-19 B cells. IA and IH also cured infected hamsters with lower toxicity than Glucantime.


Conclusions/Significance 
Immucillins IA, IH and SMIH were effective in treating leishmaniasis in mice. In hamsters, IA and IH were also effective. The highest therapeutic efficacy was obtained with IA, possibly due to its induction of a TH1 immune response. Low immucillin doses were required and showed no toxicity. Our results disclose the potential use of IA and IH in the therapy of visceral leishmaniasis.

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Immucillins ImmA and ImmH Are Effective and Non-toxic in the Treatment of Experimental Visceral Leishmaniasis

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.

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

Marcus Vinícius Alves-Silva

Papers

A Chimera Containing CD4+ and CD8+ T-Cell Epitopes of the Leishmania donovani Nucleoside Hydrolase (NH36) Optimizes Cross-Protection against Leishmania amazonesis Infection

Cross-Protective Immunity to Leishmania amazonensis is Mediated by CD4+ and CD8+ Epitopes of Leishmania donovani Nucleoside Hydrolase Terminal Domains.

Immucillins ImmA and ImmH Are Effective and Non-toxic in the Treatment of Experimental Visceral Leishmaniasis

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