Killed but metabolically active Leishmania infantum as a novel whole-cell vaccine for visceral leishmaniasis

There are currently no effective vaccines for visceral leishmaniasis, the second most deadly parasitic infection in the world. Here, we describe a novel whole-cell vaccine approach using Leishmania infantum chagasi promastigotes treated with the psoralen compound amotosalen (S-59) and low doses of UV A radiation. This treatment generates permanent, covalent DNA cross-links within parasites and results in Leishmania organisms termed killed but metabolically active (KBMA). In this report, we characterize the in vitro growth characteristics of both KBMA L. major and KBMA L. infantum chagasi. Concentrations of S-59 that generate optimally attenuated parasites were identified. Like live L. infantum chagasi, KBMA L. infantum chagasi parasites were able to initially enter liver cells in vivo after intravenous infection. However, whereas live L. infantum chagasi infection leads to hepatosplenomegaly in mice after 6 months, KBMA L. infantum chagasi parasites were undetectable in the organs of mice at this time point. In vitro, KBMA L. infantum chagasi retained the ability to enter macrophages and induce nitric oxide production. These characteristics of KBMA L. infantum chagasi correlated with the ability to prophylactically protect mice via subcutaneous vaccination at levels similar to vaccination with live, virulent organisms. Splenocytes from mice vaccinated with either live L. infantum chagasi or KBMA L. infantum chagasi displayed similar cytokine patterns in vitro. These results suggest that KBMA technology is a potentially safe and effective novel vaccine strategy against the intracellular protozoan L. infantum chagasi. This approach may represent a new method for whole-cell vaccination against other complex intracellular pathogens

Immunomodulation by imiquimod in patients with high-risk primary melanoma.

Imiquimod is a synthetic Toll-like receptor 7 (TLR7) agonist approved for the topical treatment of actinic keratoses, superficial basal cell carcinoma, and genital warts. Imiquimod leads to an 80-100% cure rate of lentigo maligna; however, studies of invasive melanoma are lacking. We conducted a pilot study to characterize the local, regional, and systemic immune responses induced by imiquimod in patients with high-risk melanoma. After treatment of the primary melanoma biopsy site with placebo or imiquimod cream, we measured immune responses in the treated skin, sentinel lymph nodes (SLNs), and peripheral blood. Treatment of primary melanomas with 5% imiquimod cream was associated with an increase in both CD4+ and CD8+ T cells in the skin, and CD4+ T cells in the SLN. Most of the CD8+ T cells in the skin were CD25 negative. We could not detect any increases in CD8+ T cells specifically recognizing HLA-A(*)0201-restricted melanoma epitopes in the peripheral blood. The findings from this small pilot study demonstrate that topical imiquimod treatment results in enhanced local and regional T-cell numbers in both the skin and SLN. Further research into TLR7 immunomodulating pathways as a basis for effective immunotherapy against melanoma in conjunction with surgery is warranted

LXR Deficiency Confers Increased Protection against Visceral Leishmania Infection in Mice

Leishmania spp. are protozoan single-cell parasites that are transmitted to humans by the bite of an infected sand fly, and can cause a wide spectrum of disease, ranging from self-healing skin lesions to potentially fatal systemic infections. Certain species of Leishmania that cause visceral (systemic) disease are a source of significant mortality worldwide. Here, we use a mouse model of visceral Leishmania infection to investigate the effect of a host gene called LXR. The LXRs have demonstrated important functions in both cholesterol regulation and inflammation. These processes, in turn, are closely related to lipid metabolism and the development of atherosclerosis. LXRs have also previously been shown to be involved in protection against other intracellular pathogens that infect macrophages, including certain bacteria. We demonstrate here that LXR is involved in susceptibility to Leishmania, as animals deficient in the LXR gene are much more resistant to infection with the parasite. We also demonstrate that macrophages lacking LXR kill parasites more readily, and make higher levels of nitric oxide (an antimicrobial mediator) and IL-1β (an inflammatory cytokine) in response to Leishmania infection. These results could have important implications in designing therapeutics against this deadly pathogen, as well as other intracellular microbial pathogens

LXR-deficient macrophages produce increased levels of NO following exposure to Leishmania and IFN-γ in vitro.

<p>A. BMDM from individual wild-type and DKO (Sv129xC57Bl/6) mice were stimulated <i>in vitro</i> with stationary-phase <i>L. chagasi/infantum</i> promastigotes for the times indicated, in the absence (left) or presence (right) of IFN-γ pre-treament. B. BMDM (left) or peritoneal macrophages (right) from wild-type (solid bars) or LXR-DKO (empty bars) mice (C57Bl/6 background) were infected with stationary-phase <i>L. chagasi/infantum</i> (with or without IFN-γ pre-treatment) for 24 hours <i>in vitro</i>. Supernatants were removed and levels of NO were quantitated by the Griess method. Error bars represent standard deviations of triplicate NO measurements. Results are representative of at least 3 independent experiments; * denotes p<0.05, ** p<0.005.</p

Gene expression in wild-type and LXR-deficient tissues and macrophages following Leishmania infection.

<p>A. Wild-type and LXRα-deficient mice were intravenously challenged with 1×10⁷ live, stationary-phase <i>L. chagasi/infantum</i> parasites, and RNA was prepared from livers and spleens 24 and 72 hours following infection. Real-time RT-PCR was performed with SYBR primers specific for LXRα and LXRβ. B. BMDM were preincubated overnight with IFN-γ, infected with stationary-phase <i>Leishmania</i> for 1 hour, washed to remove extracellular parasites, and incubated for an additional 2 hours. RNA was harvested, and levels of various genes were determined by real-time RT-PCR. Error bars represent standard deviations of replicate infections; ** denotes p<0.005. Results are representative of at least 3 independent experiments.</p

Immunomodulation by imiquimod in patients with high-risk primary melanoma.

Imiquimod is a synthetic Toll-like receptor 7 (TLR7) agonist approved for the topical treatment of actinic keratoses, superficial basal cell carcinoma, and genital warts. Imiquimod leads to an 80-100% cure rate of lentigo maligna; however, studies of invasive melanoma are lacking. We conducted a pilot study to characterize the local, regional, and systemic immune responses induced by imiquimod in patients with high-risk melanoma. After treatment of the primary melanoma biopsy site with placebo or imiquimod cream, we measured immune responses in the treated skin, sentinel lymph nodes (SLNs), and peripheral blood. Treatment of primary melanomas with 5% imiquimod cream was associated with an increase in both CD4+ and CD8+ T cells in the skin, and CD4+ T cells in the SLN. Most of the CD8+ T cells in the skin were CD25 negative. We could not detect any increases in CD8+ T cells specifically recognizing HLA-A(*)0201-restricted melanoma epitopes in the peripheral blood. The findings from this small pilot study demonstrate that topical imiquimod treatment results in enhanced local and regional T-cell numbers in both the skin and SLN. Further research into TLR7 immunomodulating pathways as a basis for effective immunotherapy against melanoma in conjunction with surgery is warranted

LXR-deficient mice display enhanced resistance to Leishmania infection of the liver and spleen.

<p>Wild-type or LXR-deficient C57Bl/6 mice (except for (B), which were the mixed Sv129xC57Bl/6 background) were challenged with 1×10⁷ live, stationary-phase <i>L. chagasi/infantum</i> parasites, and livers and spleens harvested at various time points. A–B. Parasite loads in the livers at d28 were quantitated by microscopy. Leishman-Donovan Units (LDU)  =  [(# amastigotes/# mononuclear cells) multiplied by liver weight in mg]. C–F. Parasite loads in liver (C, E) and spleen (D, F) were quantitated by qPCR of genomic DNA from wild-type (black bars) and LXRα^−/− (white bars) tissue. Levels are expressed as # parasites/µg tissue. Groups consisted of 7–9 mice/group for A–D, 5 mice/timepoint for E–F. Results are representative of at least 3 independent experiments. Error bars represent standard error; * denotes p<0.05, ** p<0.005, p-value for <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0000886#pntd-0000886-g001" target="_blank">Fig. 1D</a>  = 0.06, comparisons to wild-type.</p