Topical resiquimod protects against visceral infection with Leishmania infantum chagasi in mice.

New prevention and treatment strategies are needed for visceral leishmaniasis, particularly ones that can be deployed simply and inexpensively in areas where leishmaniasis is endemic. Synthetic molecules that activate Toll-like receptor 7 and 8 (TLR7/8) pathways have previously been demonstrated to enhance protection against cutaneous leishmaniasis. We initially sought to determine whether the TLR7/8-activating molecule resiquimod might serve as an effective vaccine adjuvant targeting visceral leishmaniasis caused by infection with Leishmania infantum chagasi. Resiquimod was topically applied to the skin of mice either prior to or after systemic infection with L. infantum chagasi, and parasite burdens were assessed. Surprisingly, topical resiquimod application alone, in the absence of vaccination, conferred robust resistance to mice against future intravenous challenge with virulent L. infantum chagasi. This protection against L. infantum chagasi infection persisted as long as 8 weeks after the final topical resiquimod treatment. In addition, in mice with existing infections, therapeutic treatment with topical resiquimod led to significantly lower visceral parasite loads. Resiquimod increased trafficking of leukocytes, including B cells, CD4(+) and CD8(+) T cells, dendritic cells, macrophages, and granulocytes, in livers and spleens, which are the key target organs of visceralizing infection. We conclude that topical resiquimod leads to systemic immune modulation and confers durable protection against visceralizing L. infantum chagasi infection, in both prophylactic and therapeutic settings. These studies support continued studies of TLR-modulating agents to determine mechanisms of protection and also provide a rationale for translational development of a critically needed, novel class of topical, preventative, and therapeutic agents for these lethal infections.

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.

Efficacy of synthetic peptides RP-1 and AA-RP-1 against Leishmania species in vitro and in vivo.

Host defense peptides are naturally occurring molecules that play essential roles in innate immunity to infection. Based on prior structure-function knowledge, we tested two synthetic peptides (RP-1 and AA-RP-1) modeled on the conserved, microbicidal α-helical domain of mammalian CXCL4 platelet kinocidins. These peptides were evaluated for efficacy against Leishmania species, the causative agents of the group of diseases known as leishmaniasis. In vitro antileishmanial activity was assessed against three distinct Leishmania strains by measuring proliferation, metabolic activity and parasite viability after exposure to various concentrations of peptides. We demonstrate that micromolar concentrations of RP-1 and AA-RP-1 caused dose-dependent growth inhibition of Leishmania promastigotes. This antileishmanial activity correlated with rapid membrane disruption, as well as with a loss of mitochondrial transmembrane potential. In addition, RP-1 and AA-RP-1 demonstrated distinct and significant in vivo antileishmanial activities in a mouse model of experimental visceral leishmaniasis after intravenous administration. These results establish efficacy of RP-1 lineage synthetic peptides against Leishmania species in vitro and after intravenous administration in vivo and provide further validation of proof of concept for the development of these and related systemic anti-infective peptides targeting pathogens that are resistant to conventional antibiotics.

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.

BACKGROUND: The liver X receptors (LXRs) are a family of nuclear receptor transcription factors that are activated by oxysterols and have defined roles in both lipid metabolism and cholesterol regulation. LXRs also affect antimicrobial responses and have anti-inflammatory effects in macrophages. As mice lacking LXRs are more susceptible to infection by intracellular bacteria Listeria monocytogenes and Mycobacterium tuberculosis, we hypothesized that LXR might also influence macrophage responses to the intracellular protozoan parasite Leishmania chagasi/infantum, a causative agent of visceral leishmaniasis. METHODS AND FINDINGS: Surprisingly, both LXRα knock-out and LXRα/LXRß double-knock-out (DKO) mice were markedly resistant to systemic L. chagasi/infantum infection compared to wild-type mice. Parasite loads in the livers and spleens of these animals were significantly lower than in wild-type mice 28 days after challenge. Bone marrow-derived macrophages from LXR-DKO mice infected with L. chagasi/infantum in vitro in the presence of IFN-γ were able to kill parasites more efficiently than wild-type macrophages. This enhanced killing by LXR-deficient macrophages correlated with higher levels of nitric oxide produced, as well as increased gene expression of IL-1ß. Additionally, LXR ligands abrogated nitric oxide production in wild-type macrophages in response to infection. CONCLUSIONS: These observations suggest that LXR-deficient mice and macrophages mount antimicrobial responses to Leishmania infection that are distinct from those mounted by wild-type mice and macrophages. Furthermore, comparison of these findings to other intracellular infection models suggests that LXR signaling pathways modulate host antimicrobial responses in a complex and pathogen-specific manner. The LXR pathway thus represents a potential therapeutic target for modulating immunity against Leishmania or other intracellular parasites.