Rajatava Basu

Kinetoplastid Membrane Protein-11 DNA Vaccination Induces Complete Protection against Both Pentavalent Antimonial-Sensitive and -Resistant Strains of Leishmania donovani That Correlates with Inducible Nitric Oxide Synthase Activity and IL-4 Generation: Evidence for Mixed Th1- and Th2-Like Responses in Visceral Leishmaniasis

The emergence of an increasing number of Leishmania donovani strains resistant to pentavalent antimonials (SbV), the first line of treatment for visceral leishmaniasis worldwide, accounts for decreasing efficacy of chemotherapeutic interventions. A kinetoplastid membrane protein-11 (KMP-11)-encoding construct protected extremely susceptible golden hamsters from both pentavalent antimony responsive (AG83) and antimony resistant (GE1F8R) virulent L. donovani challenge. All the KMP-11 DNA vaccinated hamsters continued to survive beyond 8 mo postinfection, with the majority showing sterile protection. Vaccinated hamsters showed reversal of T cell anergy with functional IL-2 generation along with vigorous specific anti-KMP-11 CTL-like response. Cytokines known to influence Th1- and Th2-like immune responses hinted toward a complex immune modulation in the presence of a mixed Th1/Th2 response in conferring protection against visceral leishmaniasis. KMP-11 DNA vaccinated hamsters were protected by a surge in IFN-γ, TNF-α, and IL-12 levels along with extreme down-regulation of IL-10. Surprisingly the prototype candidature of IL-4, known as a disease exacerbating cytokine, was found to have a positive correlation to protection. Contrary to some previous reports, inducible NO synthase was actively synthesized by macrophages of the protected hamsters with concomitant high levels of NO production. This is the first report of a vaccine conferring protection to both antimony responsive and resistant Leishmania strains reflecting several aspects of clinical visceral leishmaniasis.

The Th17 family: flexibility follows function

Discovery of the T‐helper 17 (Th17) subset heralded a major shift in T‐cell biology and immune regulation. In addition to defining a new arm of the adaptive immune response, studies of the Th17 pathway have led to a greater appreciation of the developmental flexibility, or plasticity, that is a feature of T‐cell developmental programs. Since the initial finding that differentiation of Th17 cells is promoted by transforming growth factor‐β (TGFβ), it became clear that Th17 cell development overlapped that of induced regulatory T (iTreg) cells. Subsequent findings established that Th17 cells are also unusually flexible in their late developmental programming, demonstrating substantial overlap with conventional Th1 cells through mechanisms that are just beginning to be understood but would appear to have important implications for immunoregulation at homeostasis and in immune‐mediated diseases. Herein we examine the developmental and functional features of Th17 cells in relation to iTreg cells, Th1 cells, and Th22 cells, as a basis for understanding the contributions of this pathway to host defense, immune homeostasis, and immune‐mediated disease.

IL-1 signaling modulates activation of STAT transcription factors to antagonize retinoic acid signaling and control the TH17 cell-iTreg cell balance

Interleukin 17 (IL-17)-producing helper T cells (TH17 cells) and CD4+ inducible regulatory T cells (iTreg cells) emerge from an overlapping developmental program. In the intestines, the vitamin A metabolite retinoic acid (RA) is produced at steady state and acts as an important cofactor to induce iTreg cell development while potently inhibiting TH17 cell development. Here we found that IL-1 was needed to fully override RA-mediated expression of the transcription factor Foxp3 and induce protective TH17 cell responses. By repressing expression of the negative regulator SOCS3 dependent on the transcription factor NF-κB, IL-1 increased the amplitude and duration of phosphorylation of the transcription factor STAT3 induced by TH17-polarizing cytokines, which led to an altered balance in the binding of STAT3 and STAT5 to shared consensus sequences in developing T cells. Thus, IL-1 signaling modulated STAT activation downstream of cytokine receptors differently to control the TH17 cell–iTreg cell developmental fate.

HLA Class I—Restricted T Cell Epitopes of the Kinetoplastid Membrane Protein—11 Presented by Leishmania donovani—Infected Human Macrophages

Visceral leishmaniasis is a protozoal disease caused by the intracellular parasites Leishmania donovani and L. chagasi/infantum, and it is usually deadly if not treated. To date, no vaccine exists for prophylaxis or immunotherapy, nor has it been established which effector mechanisms of the immune system are most instrumental against the parasites. Recent reports have suggested that CD8(+) T cells, in addition to CD4(+) T cells, might play major roles in the defense against infection and in the cure of the disease. To identify epitopes recognized by CD8(+) T cells that can be used for immune monitoring to investigate the role of these cells in human visceral leishmaniasis, as well as in vaccine development, we scanned the entire sequence of the leishmanial protein kinetoplastid membrane protein (kmp)-11 with overlapping nonapeptides. Thirty peptides that specifically trigger interferon- gamma secretion by human CD8(+) T cells were identified. Four T cell lines with specificities for different peptides recognize Leishmania-infected autologous macrophages, which proves that kmp-11 is processed and presented via the major histocompatibility complex class I pathway of infected cells. Kmp-11 is thus a candidate antigen for the development of T cell vaccines.

Mapping the Antigenicity of the Parasites in Leishmania donovani Infection by Proteome Serology

Background Leishmaniasis defines a cluster of protozoal diseases with diverse clinical manifestations. The visceral form caused by Leishmania donovani is the most severe. So far, no vaccines exist for visceral leishmaniasis despite indications of naturally developing immunity, and sensitive immunodiagnostics are still at early stages of development. Methodology/Principle Findings Establishing a proteome-serological methodology, we mapped the antigenicity of the parasites and the specificities of the immune responses in human leishmaniasis. Using 2-dimensional Western blot analyses with sera and parasites isolated from patients in India, we detected immune responses with widely divergent specificities for up to 330 different leishmanial antigens. 68 antigens were assigned to proteins in silver- and fluorochrome-stained gels. The antigenicity of these proteins did not correlate with the expression levels of the proteins. Although some antigens are shared among different parasite isolates, there are extensive differences and no immunodominant antigens, but indications of antigenic drift in the parasites. Six antigens were identified by mass spectrometry. Conclusions/Significance Proteomics-based dissection of the serospecificities of leishmaniasis patients provides a comprehensive inventory of the complexity and interindividual heterogeneity of the host-responses to and variations in the antigenicity of the Leishmania parasites. This information can be instrumental in the development of vaccines and new immune monitoring and diagnostic devices.

KMP-11 DNA immunization significantly protects against L. donovani infection but requires exogenous IL-12 as an adjuvant for comparable protection against L. major

As vaccine potential of cross-species protection by a candidate antigen is less explored, in this study we compared cross-specific protective efficacy of Kinetoplastid Membrane Protein-11 (KMP-11) as a DNA vaccine alone and in conjunction with exogenous IL-12 administration in experimental BALB/c model against two most widely prevalent forms of clinical diseases caused by Leishmania major (LM) and Leishmania donovani (LD). Whereas, KMP-11 DNA vaccination alone showed significant potential in terms of resolution of splenic and hepatic parasite burden against virulent LD challenge, it showed considerably less efficacy (<70% reduction) against virulent LM challenge in terms of presence of parasite in lymph node. Remarkably exogenous IL-12 administration in the form of IL-12 p35/p40 expression vectors or recombinant protein along with KMP-11 DNA had exactly opposing effect on protection against LM and LD. Exogenous IL-12 administration significantly increased residual LD-burden but enhanced the protective efficacy of KMP-11 DNA vaccine against LM compared to KMP-11 immunization alone. Elucidation of effector mechanism showed KMP-11 DNA induced protection against LD was associated with the generation of mixed Th1/Th2 response, while KMP-11/IL-12-induced comparable protection against LM was associated with high IgG2a titre indicative of a polarized Th1 response. Exogenous IL-12 administration resulted in robust gamma interferon (IFN-gamma) production and suppression of IL-4 from CD4+ T cell against both LM and LD. Nevertheless protective immune response was only compromised against LD infection where frequency of anti-KMP-11 CTL response was significantly reduced after exogenous IL-12 administration. Our study provides a comparative evaluation of effector mechanisms in the assessment of cross-specific protection by KMP-11 and KMP-11/IL-12 immunization against these two prevalent forms of leishmaniasis.

Hybrid Cell Vaccination Resolves Leishmania donovani Infection by Eliciting a Strong CD8+ Cytotoxic T-Lymphocyte Response with Concomitant Suppression of Interleukin-10 (IL-10) but Not IL-4 or IL-13

ABSTRACT There is an acute dearth of therapeutic interventions against visceral leishmaniasis that is required to restore an established defective cell-mediated immune response. Hence, formulation of effective immunotherapy requires the use of dominant antigen(s) targeted to elicit a specific antiparasitic cellular immune response. We implemented hybrid cell vaccination therapy in Leishmania donovani-infected BALB/c mice by electrofusing dominant Leishmania antigen kinetoplastid membrane protein 11 (KMP-11)-transfected bone marrow-derived macrophages from BALB/c mice with allogeneic bone marrow-derived dendritic cells from C57BL/6 mice. Hybrid cell vaccine (HCV) cleared the splenic and hepatic parasite burden, eliciting KMP-11-specific major histocompatibility complex class I-restricted CD8+ cytotoxic T-lymphocyte (CTL) responses. Moreover, splenic lymphocytes of HCV-treated mice not only showed the enhancement of gamma interferon but also marked an elevated expression of the Th2 cytokines interleukin-4 (IL-4) and IL-13 at both transcriptional and translational levels. On the other hand, IL-10 production from splenic T cells was markedly suppressed as a result of HCV therapy. CD8+ T-cell depletion completely abrogated HCV-mediated immunity and the anti-KMP-11 CTL response. Interestingly, CD8+ T-cell depletion completely abrogated HCV-induced immunity, resulting in a marked increase of IL-10 but not of IL-4 and IL-13. The present study reports the first implementation of HCV immunotherapy in an infectious disease model, establishing strong antigen-specific CTL generation as a correlate of HCV-mediated antileishmanial immunity that is reversed by in vivo CD8+ T-cell depletion of HCV-treated mice. Our findings might be extended to drug-nonresponsive visceral leishmaniasis patients, as well as against multiple infectious diseases with pathogen-specific immunodominant antigens.

Cellular and Molecular Dynamics of Th17 Differentiation and its Developmental Plasticity in the Intestinal Immune Response

After emerging from the thymus, naive CD4 T cells circulate through secondary lymphoid tissues, including gut-associated lymphoid tissue of the intestine. The activation of naïve CD4 T cells by antigen-presenting cells offering cognate antigen initiate differentiation programs that lead to the development of highly specialized T helper (Th) cell lineages. Although initially believed that developmental programing of effector T cells such as T helper 1 (Th1) or T helper 2 (Th2) resulted in irreversible commitment to a fixed fate, subsequent studies have demonstrated greater flexibility, or plasticity, in effector T cell stability than originally conceived. This is particularly so for the Th17 subset, differentiation of which is a highly dynamic process with overlapping developmental axes with inducible regulatory T (iTreg), T helper 22 (Th22), and Th1 cells. Accordingly, intermediary stages of Th17 cells are found in various tissues, which co-express lineage-specific transcription factor(s) or cytokine(s) of developmentally related CD4 T cell subsets. A highly specialized tissue like that of the intestine, which harbors the largest immune compartment of the body, adds several layers of complexity to the intricate process of Th differentiation. Due to constant exposure to millions of commensal microbes and periodic exposure to pathogens, the intestinal mucosa maintains a delicate balance between regulatory and effector T cells. It is becoming increasingly clear that equilibrium between tolerogenic and inflammatory axes is maintained in the intestine by shuttling the flexible genetic programming of a developing CD4 T cell along the developmental axis of iTreg, Th17, Th22, and Th1 subsets. Currently, Th17 plasticity remains an unresolved concern in the field of clinical research as targeting Th17 cells to cure immune-mediated disease might also target its related subsets. In this review, we discuss the expanding sphere of Th17 plasticity through its shared developmental axes with related cellular subsets such as Th22, Th1, and iTreg in the context of intestinal inflammation and also examine the molecular and epigenetic features of Th17 cells that mediate these overlapping developmental programs.

Identification of New Antigens in Visceral Leishmaniasis by Expression Cloning and Immunoblotting with Sera of Kala-Azar Patients from Bihar, India

ABSTRACT Sera of kala-azar patients from Bihar, India, were used to identify Leishmania donovani antigens encoded by a phage expression library. Ten antigens were identified, five of which have not been described as leishmania antigens before. The antigens specifically react with sera of leishmania-infected patients but not of toxoplasma- or plasmodium-infected patients.

Infectivity and attenuation of Leishmania donovani promastigotes II: Association of the loss of parasite infectivity with the terminal galactosylation of precursor acceptors present in virulent parasites by the developmentally regulated galactosyltransferase

The β1‐4 galactosyltransferase enzyme of the Leishmania donovani promastigotes, was found to be developmentally regulated and expressed only in the attenuated parasites. The enzymatic product of soluble determinants of virulent promastigotes and the galactosyltransferase enzyme was found to stimulate the macrophage burst activity but inhibit in vitro intracellular parasitism. In contrast, removal of terminal galactose moieties from soluble determinants of attenuated parasites resulted in the inhibition of macrophage respiratory burst activity but did not now inhibit intracellular parasitism. We propose that the terminal galactosylation of acceptor substrates present in virulent parasites by the developmentally regulated galactosyltransferase is associated with loss of parasite virulence.