Salvador Iborra

The DC receptor DNGR-1 mediates cross-priming of CTLs during vaccinia virus infection in mice

In order to prime T cells, DCs integrate signals emanating directly from pathogens and from their noxious action on the host. DNGR-1 (CLEC9A) is a DC-restricted receptor that detects dead cells. Therefore, we investigated the possibility that DNGR-1 affects immunity to cytopathic viruses. DNGR-1 was essential for cross-presentation of dying vaccinia virus-infected (VACV-infected) cells to CD8(+) T cells in vitro. Following injection of VACV or VACV-infected cells into mice, DNGR-1 detected the ligand in dying infected cells and mediated cross-priming of anti-VACV CD8(+) T cells. Loss of DNGR-1 impaired the CD8+ cytotoxic response to VACV, especially against those virus strains that are most dependent on cross-presentation. The decrease in total anti-VACV CTL activity was associated with a profound increase in viral load and delayed resolution of the primary lesion. In addition, lack of DNGR-1 markedly diminished protection from infection induced by vaccination with the modified vaccinia Ankara (MVA) strain. DNGR-1 thus contributes to anti-VACV immunity, following both primary infection and vaccination. The non-redundant ability of DNGR-1 to regulate cross-presentation of viral antigens suggests that this form of regulation of antiviral immunity could be exploited for vaccination.

Gene expression induced by Toll-like receptors in macrophages requires the transcription factor NFAT5

NFAT5 regulates the induction of TLR-stimulated genes with constitutive binding to the Tnf promoter regardless of TLR ligation and recruitment to Nos2 and Il6 dependent on TLR activation and IKKb.

Immunohistological features of visceral leishmaniasis in BALB/c mice

It has been reported that the level of protection provided by vaccines against murine visceral leishmaniasis (VL) is low and that progress in research on VL may be due to the lack of appropriate models to study protective immunity. We have analysed the immunohistological features occurring in BALB/c mice after intravenous administration of 10 3 , 10 5 and 10 6 parasites of Leishmania infantum. Our results show that in all cases parasite administration leads to the establishment of infection and to the development of quantifiable immunohistological features which are dependent on the inoculum size. This study demonstrates that differences in the parasite challenge result in changes in the evolution of some of the parameters associated with the degree of the infection in the BALB/c model: level of anti‐Leishmania antibodies, up‐regulation of spleen arginase activity, balance between IFN‐γ and IL‐10, extent of lymphoid follicle depletion in the splenic white pulp and ineffective development of hepatic granulomas. Also, and depending on the initial infectious inoculum, the absence of parasites in the bone marrow and the number of mature and empty type granulomas were parameters associated with protection. We think that in this model a challenge of the order of 105 parasites should prove useful for vaccine studies against VL.

Vaccination with the Leishmania infantum Acidic Ribosomal P0 Protein plus CpG Oligodeoxynucleotides Induces Protection against Cutaneous Leishmaniasis in C57BL/6 Mice but Does Not Prevent Progressive Disease in BALB/c Mice

ABSTRACT We have examined the efficacy of the administration in mice of a molecularly defined vaccine based on the Leishmania infantum acidic ribosomal protein P0 (rLiP0). Two different challenge models of murine cutaneous leishmaniasis were used: (i) subcutaneous inoculation of L. major parasites in susceptible BALB/c mice (a model widely used for vaccination analysis) and (ii) the intradermal inoculation of a low infective dose in resistant C57BL/6 mice (a model that more accurately reproduces the L. major infection in natural reservoirs and in human hosts). First, we demonstrated that C57BL/6 mice vaccinated with LiP0-DNA or rLiP0 protein plus CpG oligodeoxynucleotides (ODN) were protected against the development of dermal pathology and showed a reduction in the parasite load. This protection was associated with production of gamma interferon (IFN-γ) in the dermal site. Secondly, we showed that immunization with rLiP0 plus CpG ODN is able to induce only partial protection in BALB/c, since these mice finally developed a progressive disease. Further, we demonstrated that LiP0 vaccination induces a Th1 immunological response in both strains of mice. In both cases, the antibodies against LiP0 were predominantly of the immunoglobulin G2a isotype, which was correlated with an rLiP0-stimulated production of IFN-γ in draining lymph nodes. Finally, we demonstrated that LiP0 vaccination does not prevent the Th2 response induced by L. major infection in BALB/c mice. Taken together, these data indicate that the BALB/c model of cutaneous leishmaniasis may undervalue the potential efficacy of some vaccines based on defined proteins, making C57BL/6 a suitable alternative model to test vaccine candidates.

The Leishmania infantum acidic ribosomal protein P0 administered as a DNA vaccine confers protective immunity to Leishmania major infection in BALB/c mice.

ABSTRACT In this study, we examined the immunogenic properties of the Leishmania infantum acidic ribosomal protein P0 (LiP0) in the BALB/c mouse model. The humoral and cellular responses induced by the administration of the LiP0 antigen, either as soluble recombinant LiP0 (rLiP0) or as a plasmid DNA formulation (pcDNA3-LiP0), were determined. Also, the immunological response associated with a prime-boost strategy, consisting of immunization with pcDNA3-LiP0 followed by a boost with rLiP0, was assayed. Immunization with rLiP0 induced a predominant Th2-like humoral response, but no anti-LiP0 antibodies were induced after immunization with pcDNA3-LiP0, whereas a strong humoral response consisting of a mixed immunoglobulin G2a (IgG2a)-IgG1 isotype profile was induced in mice immunized with the prime-boost regime. For all three immunization protocols, rLiP0-stimulated production of gamma interferon (IFN-γ) in both splenocytes and lymph node cells from immunized mice was observed. However, it was only when mice were immunized with pcDNA3-LiP0 that noticeable protection against L. major infection was achieved, as determined by both lesion development and parasite burden. Immunization of mice with LiP0-DNA primes both CD4+ and CD8+ T cells, which, with the L. major challenge, were boosted to produce significant levels of IL-12-dependent, antigen-specific IFN-γ. Taken together, these data indicate that genetic vaccination with LiP0 induces protective immunological effector mechanisms, yet the immunological response elicited by LiP0 is not sufficient to keep the infection from progressing.

Vaccination with the Leishmania major ribosomal proteins plus CpG oligodeoxynucleotides induces protection against experimental cutaneous leishmaniasis in mice

In the present work we analyze the antigenicity of Leishmania major ribosomal proteins (LRP) in infected BALB/c mice. We show that BALB/c mice vaccinated with LRP in the presence of CpG oligodeoxynucleotides (CpG-ODN) were protected against the development of dermal pathology and showed a reduction in the parasite load after challenge with L. major. This protection was associated with the induction of an IL-12 dependent specific-IFN-gamma response mediated mainly by CD4(+) T cell, albeit a minor contribution of CD8(+) T cells cannot be ruled out. Induction of Th1 responses against LRP also resulted in a reversion of the Th2 responses associated with susceptibility. A marked reduction of IgG1 antibody titer against parasite antigens besides an impaired IL-4 and IL-10 cytokine production by parasite specific T cells was observed. In addition, we show that the administration of the LRP plus CpG-ODN preparation also conferred protection in the naturally resistant C57BL/6 mice. In this strain protection was associated with a LRP specific IFN-gamma production in lymph nodes draining the challenge site. We believe that these evolutionary conserved proteins, combined with adjuvants that favor Th1 responses, may be relevant components of a pan-Leishmania vaccine.

Batf3-dependent CD103+ dendritic cells are major producers of IL-12 that drive local Th1 immunity against Leishmania major infection in mice

The role of different DC subsets in priming and maintenance of immunity against Leishmania major (L. major) infection is debated. The transcription factor basic leucine zipper transcription factor, ATF‐like 3 (Batf3) is essential for the development of mouse CD103+ DCs and some functions of CD8α+ DCs. We found that CD103+ DCs were significantly reduced in the dermis of Batf3‐deficient C57BL/6 mice. Batf3−/− mice developed exacerbated and unresolved cutaneous pathology following a low dose of intradermal L. major infection in the ear pinnae. Parasite load was increased 1000‐fold locally and expanded systemically. Batf3 deficiency did not affect L. major antigen presentation to T cells, which was directly exerted by CD8α− conventional DCs (cDCs) in the skin draining LN. However, CD4+ T‐cell differentiation in the LN and skin was skewed to nonprotective Treg‐ and Th2‐cell subtypes. CD103+ DCs are major IL‐12 producers during L. major infection. Local Th1 immunity was severely hindered, correlating with impaired IL‐12 production and reduction in CD103+ DC numbers. Adoptive transfer of WT but not IL‐12p40−/− Batf3‐dependent DCs significantly improved anti‐L. major response in infected Batf3−/− mice. Our results suggest that IL‐12 production by Batf3‐dependent CD103+ DCs is crucial for maintenance of local Th1 immunity against L. major infection.

Recent advances in vaccines for leishmaniasis.

The observation that recovery from infection with Leishmania confers immunity to reinfection suggests that control of leishmaniasis by vaccination may be possible. However, there are no vaccines available at present to control any form of leishmaniasis, despite considerable efforts. Studies of the immunopathogenesis and mechanisms of protective immunity, mainly derived from animal models of experimental leishmaniasis, have defined a number of features that should be met by an effective vaccine. In addition, several antigens have been identified that may be potential vaccine candidates, and molecular biological techniques have made them available as recombinant proteins for second-generation vaccines. Furthermore, molecules present in the saliva of Leishmania-transmitting vectors have been demonstrated as valuable candidates for the development of anti-Leishmania vaccines. This review concentrates on the most promising vaccine candidates and highlights new approaches for the development of vaccines. Finally, based on present knowledge, the future prospects for developing an effective vaccine against the different clinical forms of leishmaniasis are discussed.

Furin-Processed Antigens Targeted to the Secretory Route Elicit Functional TAP1−/−CD8+ T Lymphocytes In Vivo

Most pathogen-derived peptides recognized by CD8+ CTL are produced by proteasomes and delivered to the endoplasmic reticulum by the TAP transporters associated with Ag processing. Alternative proteases also produce antigenic peptides, but their actual relevance is unclear. There is a need to quantify the contribution of these supplementary pathways in vitro and in vivo. A well-defined TAP-independent secretory route of Ag processing involves the trans-Golgi network protease furin. Quantitation of this route by using OVA constructs encoded by vaccinia viruses indicates that it provides approximately one-third of all surface complexes of peptide and MHC class I molecules. Generation of the epitope carboxyl terminus is a dramatic rate-limiting step, since bypassing it increased efficiency by at least 1000-fold. Notably, the secretory construct activated a similar percentage of Ag-specific CD8+ T cells in wild type as in TAP1-deficient mice, which allow only secretory routes but which have a 10- to 20-fold smaller CD8 compartment. Moreover, these TAP1−/− OVA-specific CD8+ T lymphocytes accomplished elimination of epitope-bearing cells in vivo. The results obtained with this experimental system underscore the potential of secretory pathways of MHC class I Ag presentation to elicit functional CD8+ T lymphocytes in vivo and support the hypothesis that noncytosolic processing mechanisms may compensate in vivo for the lack of proteasome participation in Ag processing in persons genetically deficient in TAP and thus contribute to pathogen control.

Signalling versatility following self and non-self sensing by myeloid C-type lectin receptors.

Among myeloid immune receptors, C-type lectin receptors (CLRs) have a remarkable capacity to sense a variety of self and non-self ligands. The coupling of CLRs to different signal transduction modules is influenced not only by the receptor, but also by the nature, density and architecture of the ligand, which can affect the rate of receptor internalization and trafficking to diverse intracellular compartments. Understanding how the variety of self and non-self ligands triggers differential CLR signalling and function presents a fascinating biological challenge. Non-self ligands usually promote inflammation and immunity, whereas self ligands are frequently involved in communication and tolerance. But pathogens can mimic self-inhibitory signals to escape immune surveillance, and endogenous ligands can contribute to the sensing of pathogens through CLRs. In this review, we survey the complexity and flexibility in functional outcome found in the myeloid CLRs, which is not only based on their differing intracellular motifs, but is also conditioned by the physical nature, affinity and avidity of the ligand.