Vaccination with Nucleoside Hydrolase (NH36) of L.(L.) Donovani or its C-terminal Portion (F3) in Formulation with Saponin Prevents the Increase of the Proportions of Spleen Dendritic Cells in Murine Experimental Visceral Leishmaniasis

AbstractVisceral leishmaniasis is a chronicand lethal parasite disease against which no human vaccine is available.Hepato- splenomegaly and a progressive suppression of the cellular immune response are among its most important clinical signs. The characteristic cellular immunosupression was described as being mediated in part, through the spatial segregation of dendritic cells (DCs) and T cell lymphocytes due to altered frequencies and migration capabilities of DCs. In this investigation, we measured the spleen/body relative weight, the spleen parasite load and the total counts of spleen DCs of C57BL6 mice infected with Leishmania chagasi. All the variables achieved their maximum at 30 days after infection. We detected in infected animals a 5.08 fold increase of spleen relative weight, a 19.6 fold increase of parasite load and a 4.55 increase of total DCs counts, when compared to naïve controls. We further analysed the efficacy of the NH36 and F3 vaccines formulated in saponin in prevention of visceral leishmaniasis. When compared to the infected controls, both vaccines determined strong protection. The F3 vaccine induced the highest efficacy showing 95% and 49% reduction the parasite load and splenomegaly, respectively. The NH36 vaccine, on the other hand, developed a slightly lower but still significant protection reducing by 87% the parasite load and by 39% the spleen relative weight. Both vaccines also prevented the increase in total counts of DCs with no significant difference between them (36% by the NH36 and 26% by the F3 vaccine). Our results suggest that vaccination against murine visceral leishmaniasis with the NH36 vaccine can prevent the development of the disease by preventing the DCs dysfunction-related immunosupression. Additionally, they disclose the potential use of the NH36 C-terminal moiety, the F3 peptide for optimization of the vaccine efficacy

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

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

Resistance to visceral leishmaniasis is severely compromised in mice deficient of bradykinin B2-receptors

Abstract Background Kinins liberated from plasma–borne kininogens, are potent innate stimulatory signals. We evaluated whether resistance to infection by Leishmania (L.) chagasi depends on activation of G-protein coupled bradykinin B2 receptors (B2R). Findings B2R −/− C57BL/6 knock-out (KOB2) and B2R+/+ C57BL/6-wild type control mice (C57) were infected with amastigotes of Leishmania (L.) chagasi. Thirty days after infection, the KOB2 mice showed 14% and 32% relative increases of liver (p Conclusions We found that mice lacking B2R display increased susceptibility to the infection by Leishmania (L.) chagasi. Our findings suggest that activation of the bradykinin/B2R pathway contributes to development of host resistance to visceral leishmaniasis.</p

F1 Domain of the Leishmania (Leishmania) donovani Nucleoside Hydrolase Promotes a Th1 Response in Leishmania (Leishmania) infantum Cured Patients and in Asymptomatic Individuals Living in an Endemic Area of Leishmaniasis

The Leishmania (Leishmania) donovani nucleoside hydrolase NH36 is the main antigen of the Leishmune® vaccine and one of the promising candidates for vaccination against visceral leishmaniasis. The antigenicity of the N-terminal (F1), the central (F2), or the C-terminal recombinant domain (F3) of NH36 was evaluated using peripheral blood mononuclear cells (PBMC) from individuals infected with L. (L.) infantum from an endemic area of visceral leishmaniasis of Spain. Both NH36 and F1 domains significantly increased the PBMC proliferation stimulation index of cured patients and infected asymptomatic individuals compared to healthy controls. Moreover, F1 induced a 19% higher proliferative response than NH36 in asymptomatic exposed subjects. In addition, in patients cured from visceral leishmaniasis, proliferation in response to NH36 and F1 was accompanied by a significant increase of IFN-γ and TNF-α secretion, which was 42-43% higher, in response to F1 than to NH36. The interleukin 17 (IL-17) secretion was stronger in asymptomatic subjects, in response to F1, as well as in cured cutaneous leishmaniasis after NH36 stimulation. While no IL-10 secretion was determined by F1, a granzyme B increase was detected in supernatants from cured patients after stimulation with either NH36 or F1. These data demonstrate that F1 is the domain of NH36 that induces a recall cellular response in individuals with acquired resistance to the infection by L. (L.) infantum. In addition, F1 and NH36 discriminated the IgG3 humoral response in patients with active visceral leishmaniasis due to L. (L.) donovani (Ethiopia) and L. (L.) infantum (Spain) from that of endemic and non-endemic area controls. NH36 showed higher reactivity with sera from L. (L.) donovani-infected individuals, indicating species specificity. We conclude that the F1 domain, previously characterized as an inducer of the Th1 and Th17 responses in cured/exposed patients infected with L. (L.) infantum chagasi, may also be involved in the generation of a protective response against L. (L.) infantum and represents a potential vaccine candidate for the control of human leishmaniasis alone, or in combination with other HLA epitopes/antigens.This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPQ) fellowships 300639/2003-1 to MP, 310977/2014-2 to CP-d-S, and grant 404400/2012-4 to CP-d-S, MP, PL, RA, JM, EC); Fundação Carlos Chagas de Amparo à Pesquisa do Estado de Rio de Janeiro (FAPERJ) (grant E-26-201.583/2014, E-26-102957/2011, and E-26/111.682/2013 to CP-d-S and fellowships E-26/102415/2010 and E-26/201747/2015 to DN); Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (grant 23038.005304/2011-0 to MS); CNPQ-Fundação de Apoio à Pesquisa e a Inovação Tecnológica do Estado de Sergipe-PRONEX (12/2009); and FAPITEC CNPq-(PRONEX)(019.203.02712/2009-8) to RA. EC was supported by a research contract funded via VII PN I+D+I 2013–2016 and FEDER Funds (RICET RD12/0018/0003).S

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

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

The adjuvanticity of Chiococca alba saponins increases with the length and hydrophilicity of their sugar chains

The saponins of Chiococca alba are triterpene bidesmosides that contain glycidic moieties attached to the C-3 and C-28 carbon of their aglycone. We describe that their adjuvant potential increases in direct relationship to the length and hydrophilicity of the C-28 attached sugar chain which contains: arabinose-rhamnose in the CA2, arabinose-rhamnose-xylose in the CA3X; arabinose-rhamnose-apiose in the CA3 and arabinose-rhamnose-apiose-apiose in the CA4 saponin. the hydrophile/lipophile balance calculated for CA2 was 12.7, for CA3 and CA3X was 15.8 and for CA4 19.9. All saponins were formulated with the FML antigen for mice prophylaxis against visceral leishmaniasis. the immune response was studied using an ELISA-antibody assay and monitoring of the intradermal response (IDR) to Leishmania antigens, the cytokine expression in supernatants and the intracellular staining of in vitro cultured splenocytes. After challenge, significant increases of IgG and IgG2a antibodies were noted only in the CA4 vaccinated mice that showed extended IDR, higher IFN-gamma production by CD8+ and TNF-alpha production by CD4+ T cells, higher TNF-alpha secretion and the highest reduction of the parasite load (78%). the increases in IDR, CD4-TNF-alpha,CD8-IFN-gamma and CD8-TNF-alpha by the CA4 vaccine were strong correlates of protection and were significantly correlated to the decrease of parasite load (p=-0.007). Protection generated by the CA4 vaccine was mainly mediated by a CD4+ T cell and a TNF-alpha driven response with a lower contribution of CD8+ T cells, as confirmed by an in vivo depletion with monoclonal antibodies and by vaccination assays in TNF-alpha-receptor knock-out mice. Our results confirm that the superiority of the CA4 saponin is related to the higher hydrophilicity of its longer carbohydrate chain. C. alba saponins were non-toxic and only the xylose-containing saponin CA3X was hemolytic (HD50 = 87 mu g/ml). the increase in sugar units of the saponins is positively correlated to the increase of IDR and to the decrease of parasite load. (C) 2012 Elsevier B.V. All rights reserved.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, CCS, Ilha Fundao,Dept Microbiol Geral, BR-21941902 Rio de Janeiro, BrazilUniv Fed Rio de Janeiro, Nucleo Pesquisas Prod Nat, BR-21941902 Rio de Janeiro, BrazilUFRJ, Hosp Univ Clementino Fraga Filho, Fac Med, BR-21941913 Rio de Janeiro, BrazilUniversidade Federal de São Paulo UNIFESP, Ctr Interdisciplinar Terapia Genica, BR-04044010 São Paulo, BrazilUniversidade Federal de São Paulo UNIFESP, Ctr Interdisciplinar Terapia Genica, BR-04044010 São Paulo, BrazilCNPq: 500992/2008-8FAPERJ: E-26/110305/2007FAPERJ: E-26/110132/2007FAPERJ: E-26/100416/2007FAPERJ: E-22/102733/2008Web of Scienc

Vaccination, challenge and development of NH36-specific humoral immune response.

<p>(<b>A</b>) Study design: Balb/c mice were vaccinated with NH36sap, F1sap, F2sap or F3sap at the indicated time intervals, through the sc route, followed by intravenous challenge with <i>L. chagasi</i> amastigotes<b>.</b> Bars represent the mean ± SE of the absorbance values of anti-NH36 antibodies from 1/100 diluted serum of two independent experiments (n = 11–12 mice per treatment) after immunization (<b>B</b>) and after challenge (<b>C</b>). <b>*</b> p<0.05 different from the saline control. p<0.05 different from F1sap vaccine; <b>○</b> p<0.05 different from the F2sap vaccine; ◆ p<0.05 different from NH36sap vaccine; p<0.05 different from all other vaccines.</p

Nucleoside hydrolase NH36 T cell and antibody epitope mapping.

<p>The peptide sequence of MHC class II-IA^d and IE^d, haplotype H2^d CD4+ T cell epitopes (bold), of MHC class I L^d-CD8+ T cell predicted epitopes (underlined) and of epitopes for antibodies (grey background) in the F1, F2 and F3 fragments of the NH36 Nucleoside hydrolase of <i>Leishmania donovani</i>.</p