Leishmaniavirus-dependent metastatic leishmaniasis is prevented by blocking IL-17A

Cutaneous leishmaniasis has various outcomes, ranging from self-healing reddened papules to extensive open ulcerations that metastasise to secondary sites and are often resistant to standard therapies. In the case of L. guyanensis (L.g), about 5-10% of all infections result in metastatic complications. We recently showed that a cytoplasmic virus within L.g parasites (LRV1) is able to act as a potent innate immunogen, worsening disease outcome in a murine model. In this study, we investigated the immunophenotype of human patients infected by L.g and found a significant association between the inflammatory cytokine IL-17A, the presence of LRV1 and disease chronicity. Further, IL-17A was inversely correlated to the protective cytokine IFN-γ. These findings were experimentally corroborated in our murine model, where IL-17A produced in LRV1+ L.g infection contributed to parasite virulence and dissemination in the absence of IFN-γ. Additionally, IL-17A inhibition in mice using digoxin or SR1001, showed therapeutic promise in limiting parasite virulence. Thus, this murine model of LRV1-dependent infectious metastasis validated markers of disease chronicity in humans and elucidated the immunologic mechanism for the dissemination of Leishmania parasites to secondary sites. Moreover, it confirms the prognostic value of LRV1 and IL-17A detection to prevent metastatic leishmaniasis in human patients

Severe Cutaneous Leishmaniasis in a Human Immunodeficiency Virus Patient Coinfected with Leishmania braziliensis and Its Endosymbiotic Virus.

Leishmania parasites cause a broad range of disease, with cutaneous afflictions being, by far, the most prevalent. Variations in disease severity and symptomatic spectrum are mostly associated to parasite species. One risk factor for the severity and emergence of leishmaniasis is immunosuppression, usually arising by coinfection of the patient with human immunodeficiency virus (HIV). Interestingly, several species of Leishmania have been shown to bear an endogenous cytoplasmic dsRNA virus (LRV) of the Totiviridae family, and recently we correlated the presence of LRV1 within Leishmania parasites to an exacerbation murine leishmaniasis and with an elevated frequency of drug treatment failures in humans. This raises the possibility of further exacerbation of leishmaniasis in the presence of both viruses, and here we report a case of cutaneous leishmaniasis caused by Leishmania braziliensis bearing LRV1 with aggressive pathogenesis in an HIV patient. LRV1 was isolated and partially sequenced from skin and nasal lesions. Genetic identity of both sequences reinforced the assumption that nasal parasites originate from primary skin lesions. Surprisingly, combined antiretroviral therapy did not impact the devolution of Leishmania infection. The Leishmania infection was successfully treated through administration of liposomal amphotericin B

Exacerbated leishmaniasis caused by a viral endosymbiont can be prevented by immunization with Its viral capsid

Recent studies have shown that a cytoplasmic virus called Leishmaniavirus (LRV) is present in some Leishmania species and acts as a potent innate immunogen, aggravating lesional inflammation and development in mice. In humans, the presence of LRV in Leishmania guyanensis and in L. braziliensis was significantly correlated with poor treatment response and symptomatic relapse. So far, no clinical effort has used LRV for prophylactic purposes. In this context, we designed an original vaccine strategy that targeted LRV nested in Leishmania parasites to prevent virus-related complications. To this end, C57BL/6 mice were immunized with a recombinant LRV1 Leishmania guyanensis viral capsid polypeptide formulated with a T helper 1-polarizing adjuvant. LRV1-vaccinated mice had significant reduction in lesion size and parasite load when subsequently challenged with LRV1+ Leishmania guyanensis parasites. The protection conferred by this immunization could be reproduced in naïve mice via T-cell transfer from vaccinated mice but not by serum transfer. The induction of LRV1 specific T cells secreting IFN-γ was confirmed in vaccinated mice and provided strong evidence that LRV1-specific protection arose via a cell mediated immune response against the LRV1 capsid. Our studies suggest that immunization with LRV1 capsid could be of a preventive benefit in mitigating the elevated pathology associated with LRV1 bearing Leishmania infections and possibly avoiding symptomatic relapses after an initial treatment. This novel anti-endosymbiotic vaccine strategy could be exploited to control other infectious diseases, as similar viral infections are largely prevalent across pathogenic pathogens and could consequently open new vaccine opportunities

Detection of Leishmania RNA virus in Leishmania parasites.

BACKGROUND: Patients suffering from cutaneous leishmaniasis (CL) caused by New World Leishmania (Viannia) species are at high risk of developing mucosal (ML) or disseminated cutaneous leishmaniasis (DCL). After the formation of a primary skin lesion at the site of the bite by a Leishmania-infected sand fly, the infection can disseminate to form secondary lesions. This metastatic phenotype causes significant morbidity and is often associated with a hyper-inflammatory immune response leading to the destruction of nasopharyngeal tissues in ML, and appearance of nodules or numerous ulcerated skin lesions in DCL. Recently, we connected this aggressive phenotype to the presence of Leishmania RNA virus (LRV) in strains of L. guyanensis, showing that LRV is responsible for elevated parasitaemia, destructive hyper-inflammation and an overall exacerbation of the disease. Further studies of this relationship and the distribution of LRVs in other Leishmania strains and species would benefit from improved methods of viral detection and quantitation, especially ones not dependent on prior knowledge of the viral sequence as LRVs show significant evolutionary divergence. METHODOLOGY/PRINCIPAL FINDINGS: This study reports various techniques, among which, the use of an anti-dsRNA monoclonal antibody (J2) stands out for its specific and quantitative recognition of dsRNA in a sequence-independent fashion. Applications of J2 include immunofluorescence, ELISA and dot blot: techniques complementing an arsenal of other detection tools, such as nucleic acid purification and quantitative real-time-PCR. We evaluate each method as well as demonstrate a successful LRV detection by the J2 antibody in several parasite strains, a freshly isolated patient sample and lesion biopsies of infected mice. CONCLUSIONS/SIGNIFICANCE: We propose that refinements of these methods could be transferred to the field for use as a diagnostic tool in detecting the presence of LRV, and potentially assessing the LRV-related risk of complications in cutaneous leishmaniasis

LRV1 induces IL-17A secretion in murine leishmaniasis and contributes to LRV1-mediated disease severity.

<p>Mice deficient in IL-17A or TLR3 and their WT controls (C57BL/6) were infected in the hind footpads with 3x10⁶ of either LRV1+ or LRV1-<i>L</i>.<i>g</i> stationary-phase promastigotes. At the peak of infection (4 weeks post-inoculation), cells from the footpad lesions and popliteal LNs were re-stimulated <i>ex vivo</i>. IL-17A secretion in WT mice was quantified in the supernatant by ELISA in <b>(A)</b> intra-lesional biopsies and <b>(B)</b> popliteal LNs. <b>(C)</b> Change in footpad swelling in TLR3^-/- and WT mice was measured weekly as a proxy for disease score. <b>(D)</b> IL-17A secretion from popliteal LN cells was also compared between WT and TLR3^-/- mice. <b>(E)</b> Change in footpad swelling in IL-17A^-/- and WT mice was measured weekly as a proxy for disease score. Statistical analysis is indicated in black for comparison between WT and IL-17A^-/- LRV1+ infected mice, and in grey for comparison between IL-17A^-/- LRV+ and LRV- infected mice. <b>(F)</b> At the peak of infection (week 4), the parasite burden of the mice depicted in <b>(E)</b> was quantified by <i>in vivo</i> parasite luminescence, after injecting mice intra-peritoneally with luciferin. As previously for IL-17A, IFN-γ secretion in WT mice was quantified in the supernatant of restimulated cells by ELISA in <b>(G)</b> intra-lesional biopsies and <b>(H)</b> popliteal LNs. Graphs are representative of a minimum of 3 independent experiments, using at least 5 mice per condition and presented as mean ± SEM. Significance tested by an unpaired, parametric <i>t</i>-test (bar graphs) or one-way Anova (disease score), and indicated as *: P<0.05, **P<0.005, ***P<0.0001.</p

Screening for LRV in freshly-isolated human <i>L. braziliensis</i>.

<p><b>A.</b> Dot blot analysis of two parasite samples obtained from separate lesion biopsies in an infected patient: <i>Lb</i> 2169 and <i>Lb</i> 2192. Live parasites (1 to 4 µg total proteins) were spotted on a nitrocellulose membrane for LRV dsRNA detection by dot blot (J2 antibody). <i>Lg</i> M4147 LRV^high and LRV^neg were used as positive and negative controls. Upper panel: dsRNA detection by dot blot (J2). Lower panel: verification of protein quantity by Ponceau staining. <b>B.</b> J2 anti-dsRNA analysis of <i>Lb</i> 2169 by fluorescence microscopy. Green: dsRNA (J2 Ab). Blue: DAPI. <b>C.</b> Isolation of viral genomic dsRNA from the <i>Lb</i> 2169 strain. Intact and DNase-digested total nucleic acids from <i>Lb</i> 2169 parasites and <i>Lg</i> M4147 LRV^high as a control, were analyzed by gel electrophoresis (similarly to <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002006#pntd-0002006-g001" target="_blank">Figure 1A</a>). Note: with high resolution gels such as presented here (in contrast to <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002006#pntd-0002006-g001" target="_blank">Figure 1</a>), the viral genome often appears as a doublet.</p