Vaccination with Leishmania soluble antigen and immunostimulatory oligodeoxynucleotides induces specific immunity and protection against Leishmania donovani infection

In this report, we investigated the effect of ODN containing immunostimulatory CG motifs as adjuvant with soluble antigen (SA) from Leishmania donovani. BALB/c mice were vaccinated with the soluble antigen with or without CpG-ODN as adjuvant and then challenged with L. donovani metacyclic promastigotes. CpG-ODN alone resulted in partial protection against challenge with L. donovani. Immunization of mice with SA and CpG-ODN showed enhanced reduction in parasite load (~60%) when compared to SA (~40%) immunized mice. Immunization with SA by itself resulted in a mixed Th1/Th2 response whereas co-administration of SA with CpG-ODN resulted in a strong Th1 promoting isotype as they together promoted production of immunoglobulin G2a. Leishmania-specific Th1 cytokine response was induced by co-administering CpG-ODN and SA as they together promoted production of IFN-γ and IL-12. In the present study, we demonstrate that immunostimulatory phosphorothioate-modified ODN are promising immune enhancers for vaccination against visceral leishmaniaisis

Leishmania lipophosphoglycan activates the transcription factor activating protein 1 in J774A.1 macrophages through the extracellular signal-related kinase (ERK) and p38 mitogen-activated protein kinase

Leishmania donovani is an obligatory intracellular pathogen that resides and multiplies in the phagolysosomes of macrophages. The outcome of this infection depends on the balance between the host ability to activate macrophage killing and the parasite ability to suppress or evade this host immune response. Lipophosphoglycan (LPG) glycoconjugate, the surface molecule of the protozoan parasite is a virulence determinant and a major parasite molecule involved in this process. In this study, we examined the ability of Leishmania and its surface molecule, lipophosphoglycan to activate activating protein 1 (AP-1) through the mitogen-activated protein kinase (MAPK) cascade. We report here that the Leishmania surface molecule, lipophosphoglycan stimulates the simultaneous activation of all three classes of MAP kinases, extracellular signal-related kinases (ERKs), the c-jun amino-terminal kinase (JNK) and the p38 MAP kinase with differential kinetics in J774A.1 macrophage cell line. Furthermore, both L. donovani and its surface molecule lipophosphoglycan resulted in a dose- and time-dependent induction of AP-1 DNA-binding activity. We have also shown a dose-dependent increase of AP-1 binding activity in both low and high virulent strains of parasite. The use of inhibitors selective for ERK (PD98059) and p38 (SB203580) pathway showed that pre-incubation of cells with either SB203580 or PD98059 affected the binding activity of AP-1 suggesting that both p38 and ERK MAP kinase activation appear to be necessary for AP-1 activation by LPG. Lipophosphoglycan induced IL-12 production and generation of nitric oxide in murine macrophages. These results demonstrate that L. donovani LPG activates pro-inflammatory, endotoxin-like response pathway in J774A.1 macrophages and the interaction may play a pivotal role in the elimination of the parasite

Cholesterol is required for Leishmania donovani infection: implications in leishmaniasis

Leishmania donovani is an obligate intracellular parasite that infects macrophages of the vertebrate host, resulting in visceral leishmaniasis in humans, which is usually fatal if untreated. The molecular mechanisms involved in host-parasite interaction leading to attachment on the cell surface and subsequent internalization of the parasite are poorly characterized. Cholesterol is a major constituent of eukaryotic membranes and plays a crucial role in cellular membrane organization, dynamics, function, and sorting. It is often found distributed non-randomly in domains in membranes. Recent observations suggest that cholesterol exerts many of its actions by maintaining a specialized type of membrane domain, termed "lipid rafts", in a functional state. Lipid rafts are enriched in cholesterol and sphingolipids, and have been thought to act as platforms through which signal transduction events are coordinated and pathogens gain entry to infect host cells. We report here that cholesterol depletion from macrophage plasma membranes using methyl-β -cyclodextrin (Mβ CD) results in a significant reduction in the extent of leishmanial infection. Furthermore, the reduction in the ability of the parasite to infect host macrophages can be reversed upon replenishment of cell membrane cholesterol. Interestingly, these effects were not observed when parasites were serum-opsonized, indicating a specific requirement of cholesterol to mediate entry via the non-opsonic pathway. Importantly, we show that entry of Escherichia coli remains unaffected by cholesterol depletion. Our results therefore point to the specific requirement of plasma membrane cholesterol in efficient attachment and internalization of the parasite to macrophage cells leading to a productive infection. More importantly, these results are significant in developing novel therapeutic strategies to tackle Leishmaniasis

The sterol-binding antibiotic nystatin inhibits entry of non-opsonized Leishmania donovani into macrophages

Leishmania donovani is an obligate intracellular parasite that infects macrophages of the vertebrate host resulting in visceral leishmaniasis in humans, a major public health problem worldwide. The molecular mechanisms involved in internalization of Leishmania are still poorly characterized. We report here that cholesterol sequestration by the sterol-binding antifungal polyene antibiotic nystatin markedly inhibits binding and entry of non-opsonized L. donovani promastigotes into macrophages. Interestingly, these effects are not observed when serum-opsonized L. donovani are used for infectivity studies thus pointing the essential role of cholesterol in mediating entry of the parasite via the non-opsonic pathway. Based on our earlier results where leishmanial infectivity was shown to be sensitive to physical depletion of cholesterol from macrophages, these results indicate that the mere sequestration of cholesterol in the host plasma membrane is sufficient to inhibit the binding and entry of non-opsonized L. donovani. These results represent the first report on the effect of a cholesterol-sequestering agent on the entry of Leishmania parasites to host macrophages. More importantly, these findings offer the possibility of reevaluating the mechanism behind the effectiveness of current therapeutic strategies to treat leishmaniasis

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Immunostimulatory oligodeoxynucleotides are potent enhancers of protective immunity in mice immunized with recombinant ORFF leishmanial antigen

Unmethylated CpG dinucleotides in bacterial DNA or synthetic oligonucleotides (ODN) have proved as promising adjuvants for promotion of T helper 1 (Th1) type immune response. The potent Th1 like immune activation by CpG-ODNs suggests a possible utility for vaccination against leishmaniasis. We therefore investigated the effect of ODN containing immunostimulatory CG motifs as adjuvant with recombinant ORFF (rORFF) leishmanial antigen. BALB/c mice were vaccinated with the rORFF with or without CpG-ODN as adjuvant and then challenged with Leishmania donovani metacyclic promastigotes. Administration of CpG-ODN alone resulted in partial protection against challenge with L. donovani in BALB/c mice. Combination of rORFF and CpG-ODN showed enhanced reduction in parasite load (84%) when compared to rORFF (56%) vaccinated mice. Immunization with rORFF alone did not induce the typical Th response whereas co-administration of rORFF with CpG-ODN resulted in enhanced production of immunoglobulin G2a and interferon gamma. Our results further demonstrate that CpG-ODN alone or in combination with rORFF resulted in a dose dependent increase of nitric oxide production in activated macrophages. These studies suggest that CpG-ODN are promising immune enhancers for vaccination against visceral leishmaniasis

Vaccination with DNA encoding ORFF antigen confers protective immunity in mice infected with Leishmania donovani

The gene ORFF is part of the multigenic LD1 locus on chromosome 35 that is frequently amplified in Leishmania. The function of ORFF is unknown. The gene encoding ORFF was cloned into a eukaryotic expression vector downstream to the cytomegalovirus (CMV) promoter. BALB/c mice were injected intramuscularly with ORFF DNA and challenged with Leishmania donovani promastigotes. Vaccination with ORFF gene induced both humoral and cellular immune response against ORFF, which provided significant level of protection against challenge with L. donovani. A qualitative PCR was used to determine whether activation of Th1 cells develops selectively in response to this ORFF DNA vaccine. The results indicated that mRNA for IFN-γ was significantly induced in immunized mice. No significant change in IL-4 mRNA expression was observed in mice immunized with ORFF DNA vaccine versus mice immunized with control plasmid. Thus, DNA immunization may offer an attractive alternative strategy against leishmaniasis

A heterologous prime‐boost vaccination regimen using ORFF DNA and recombinant ORFF protein confers protective immunity against experimental visceral leishmaniasis

Objective: We describe the effectiveness of a prime-boost vaccination regimen using the open-reading frame (ORFF) gene from the LD1 locus of Leishmania donovani. Methods: A group of BALE/c mice was immunized with the plasmid carrying the gene for ORFF (F/pcDNA 3.1) and given a booster dose of either the same DNA vaccine or a vaccine with a recombinant ORFF (rORFF) protein. Another group of BALE/c mice was immunized and given a booster dose of the rORFF protein vaccine. The protective efficacies of these vaccine formulations were compared after challenge with L. donovani stationary-phase promastigotes. Results: Mice given the prime-boost vaccination regimen had an enhanced reduction in parasite load (75%-80%), compared with that in mice given only the rORFF protein vaccine (45%-60%). However, the protective response induced in the prime-boost group was not more than that elicited in the DNA vaccine group. Immunization with only the rORFF protein vaccine did not induce the typical T helper response, whereas priming with the DNA vaccine resulted in enhanced production of immunoglobulin G2a and interferon-γ. Furthermore, priming with the DNA vaccine also led to enhanced proliferation of splenocytes, suggesting subsequent expansion of antigen-specific T cells. Conclusions: The heterologous prime-boost vaccination strategy may be utilized for visceral leishmaniasis

17β-Hydroxywithanolides as Sensitizers of Renal Carcinoma Cells to Tumor Necrosis Factor‑α Related Apoptosis Inducing Ligand (TRAIL) Mediated Apoptosis: Structure–Activity Relationships

Renal cell carcinoma (RCC) is a cancer with poor prognosis, and the 5-year survival rate of patients with metastatic RCC is 5–10%. Consequently, treatment of metastatic RCC represents an unmet clinical need. Screening of a 50 000-member library of natural and synthetic compounds for sensitizers of RCC cells to TRAIL-mediated apoptosis led to identification of the 17β-hydroxywithanolide (17-BHW), withanolide E (<b>1</b>), as a promising lead. To explore structure–activity relationships, we obtained natural and semisynthetic withanolides <b>1</b>, <b>2a</b>, <b>2c</b>, and <b>3</b>–<b>36</b> and compared their ability to sensitize TRAIL-mediated apoptosis in a panel of renal carcinoma cells. Our findings revealed that 17-BHWs with a α-oriented side chain are superior to known TRAIL-sensitizing withanolides belonging to withaferin A class with a β-oriented side chain and demonstrated that the 17-BHW scaffold can be modified to enhance sensitization of RCCs to TRAIL-mediated apoptosis, thereby assisting development of natural-product-inspired drugs to treat metastatic RCC