Biology subjects

Volpedo, G.

Publications and source records attributed to Volpedo, G..

6 recordsLinked to original sources

bioRxiv2022

Leishmania major centrin knock-out parasites alter the Kynurenine- aryl hydrocarbon receptor signaling to produce a pro-inflammatory response

Leishmaniasis is a parasitic disease that is prevalent in approximately 88 countries, and yet no licensed human vaccine exists against it. Towards control of leishmaniasis, we have developed Leishmania major centrin gene deletion mutant strains (LmCen-/-) as a live attenuated vaccine, which induces a strong Th1 response to provide IFN-{gamma}-mediated protection to the host. However, the immune mechanisms of such protection remain to be understood. Metabolomic reprogramming of the host cells following Leishmania-infection has been shown to play a critical role in pathogenicity and shaping the immune response following infection. Here, we applied untargeted mass spectrometric analysis to study the metabolic changes induced by infection with LmCen-/- and compared those with virulent L. major parasite infection to identify the immune mechanism of protection. Our data shows that immunization with LmCen-/- parasites, in contrast to virulent L. major infection, alters tryptophan metabolism to down-regulate kynurenine-AhR signaling and promote a pro-inflammatory response.

systems biology↗

bioRxiv2022

Leishmania mexicana Centrin Knock out Parasites Promote M1-polarizing Metabolic Changes

Leishmaniasis is a tropical disease present in more than 90 countries. Presently, there is no approved vaccine for human use. We have previously developed live attenuated L. mexicana Cen-/- (LmexCen-/-) as a vaccine candidate that showed excellent efficacy that was characterized by reduced activation of Th2 responses and enhanced Th1 responses, contrary to wild type L. mexicana (LmexWT) infection. Towards understanding the interplay between immune mechanisms of protection and metabolic reprogramming, we applied untargeted mass spectrometric analysis to LmexCen-/- and compared them with LmexWT infection. Data showed that enriched pentose phosphate pathway (PPP) in ears immunized with LmexCen-/- parasites, compared to naive and LmexWT-infected ears. This pathway is known to promote an M1 phenotype in macrophages, suggesting a switch to a pro-inflammatory phenotype following LmexCen-/- inoculation. Accordingly, inhibition of the PPP in macrophages cultured with LmexCen-/- parasites led to diminished production of nitric oxide, IL-12, and IL-1{beta}, hallmarks of classical activation. Overall, our study revealed novel immune regulatory mechanisms that may be critical for the induction of protective immunity.

microbiology↗

bioRxiv2022

Leishmania mexicana Promotes Pain-reducing Metabolomic Reprogramming In Cutaneous Lesions

Cutaneous leishmaniasis (CL) is characterized by extensive skin lesions associated with an aggressive inflammatory reaction. Despite the extensive inflammation, CL lesions are usually painless, indicating that Leishmania infection may trigger anti-nociceptive activities in the infected tissues. To this date, the molecular mechanisms responsible for this clinical phenomenon have not been identified. Through an untargeted metabolomic analysis by mass spectrometry, we found enriched anti-nociceptive metabolic pathways in mice infected with Leishmania (L.) mexicana. In particular, endogenous purines were elevated at the lesion site during chronic infection, as well as in vitro in infected macrophages, compared to non-infected mice. These purines have known anti-inflammatory and analgesic properties by acting through adenosine receptors and inhibiting transient receptor potential channels of the vanilloid subtype 1 (TRPV1). Additionally, purine metabolites can promote interleukin (IL)-10 production, with a subsequent decrease in inflammation and pain sensitivity. We also found arachidonic acid metabolism enriched in the ear lesions compared to the non-infected controls. Arachidonic acid is a metabolite of anandamide (AEA) and 2-arachidonoylglycerol (2-AG). These endocannabinoids act on cannabinoid receptors 1 and 2 and TRPV1 channels to exert anti-inflammatory and analgesic effects. Our study provides the first evidence of metabolic pathways upregulated during L. mexicana infection that may mediate anti-nociceptive effects experienced by CL patients and identifies macrophages as a source of these metabolites. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=170 SRC="FIGDIR/small/503319v1_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@576ef2org.highwire.dtl.DTLVardef@34d939org.highwire.dtl.DTLVardef@1a13478org.highwire.dtl.DTLVardef@7bcab6_HPS_FORMAT_FIGEXP M_FIG L. mexicana infection promotes the production of purines, as well as endocannabinoid mediators, which could act on different channels of dorsal root ganglia neuron to inhibit nociception. C_FIG

microbiology↗

bioRxiv2022

Centrin-deficient Leishmania mexicana confers protection against Old World visceral leishmaniasis

Leishmaniasis is one of the top neglected tropical diseases with significant morbidity and mortality in low and middle-income countries (LMIC). However, this disease is also spreading in the developed world. Currently, there is a lack of effective strategies to control this disease. Vaccination can be an effective measure to control leishmaniasis and has the potential to achieve disease elimination. Recently, we have generated centrin gene-deleted new world L. mexicana (LmexCen-/-) parasites using CRISPR/Cas9 and showed that they protect mice against a homologous L. mexicana infection that causes cutaneous disease. In this study, we tested whether LmexCen-/- parasites can also protect against visceral leishmaniasis caused by L. donovani in a hamster model. We show that immunization with LmexCen-/- parasites is safe and does not cause lesions. Furthermore, such immunization conferred protection against visceral leishmaniasis caused by a needle-initiated L. donovani challenge, as indicated by a significant reduction in the parasite burdens in the spleen and liver and lack of mortality. Similar control of parasite burden was also observed against a sand fly mediated L. donovani challenge. Importantly, immunization with LmexCen-/- down-regulated the Th2 response as indicated by a significant reduction in the anti-inflammatory cytokines such as IL-10 and IL-4 and increased pro-inflammatory cytokine IFN-{gamma} resulting in higher IFN-{gamma}/IL-10 and IFN-{gamma}/IL4 ratios compared to non-immunized animals. This contrasts with our studies with L. major centrin deletion mutants that showed a dominant Th1 response compared to L. major wild-type infection suggesting the divergent mechanisms of protection in the two mutant parasites. LmexCen-/- immunization resulted in long-lasting protection against L. donovani infection. Further, since the efficacy of LmCen-/- has not been determined against Leishmania strains prevalent in the Americas, LmexCen-/- may be a viable alternative. Taken together, our study demonstrates that immunization with LmexCen-/- parasites is safe and efficacious against old world visceral leishmaniasis.

immunology↗

bioRxiv2020

Preclinical validation of a second generation leishmanization vaccine against vector transmitted fatal visceral leishmaniasis

Visceral Leishmaniasis (VL) is fatal if untreated. There is no licensed vaccine available against human leishmaniasis. We recently demonstrated protection in mice against L. major infection using a CRISPR genome edited attenuated Leishmania major strain (LmCen-/-). Here, as a pre-clinical step, we evaluated the protective efficacy of LmCen-/- against VL induced by sand fly transmitted Leishmania donovani in hamsters. Intradermal immunization of hamsters with LmCen-/- did not develop any lesion; while still priming a pro-inflammatory immune response. When challenged with L. donovani either by intradermal needle injection or by infected sand flies, LmCen-/--immunized hamsters were protected, not showing spleen or liver pathology averting VL fatality compared to control animals. Spleen cells from LmCen-/- immunized and infected sand fly challenged hamsters produced significantly higher Th1-associated cytokines and chemokines including IFN-{gamma} and TNF-, and significantly reduced expression of the anti-inflammatory cytokines IL-10 and IL-21, compared to non-immunized challenged animals. We further developed a GLP-grade LmCen-/- which showed equal protection as laboratory-grade LmCen-/- parasites in hamsters. Importantly, GLP-grade LmCen-/- parasites also induced a proinflammatory immune response in the PBMCs isolated from healthy people living in non-endemic and endemic for VL as well as cured VL people living in endemic region. Together, this study demonstrates that the LmCen-/- parasites are safe and efficacious against VL and it is a strong candidate vaccine to be tested in a human clinical trial.

immunology↗

bioRxiv2020

A Second Generation Leishmanization Vaccine with a Markerless Attenuated Leishmania major Strain using CRISPR gene editing

Leishmaniasis is a debilitating and often fatal neglected tropical disease caused by Leishmania protozoa transmitted by infected sand flies. Vaccination through leishmanization with live Leishmania major has been used successfully but is no longer practiced because it resulted in unacceptable skin lesions. A second generation leishmanization is described here using a CRISPR genome edited L. major strain (LmCen-/-). Notably, LmCen-/- is the first genetically engineered gene deleted Leishmania strain that is antibiotic resistant marker free and does not have any off-target mutations. Mice immunized with LmCen-/- had virtually no visible lesions following challenge with L. major-infected sand flies while non-immunized animals developed large and progressive lesions with a 2-log fold higher parasite burden. LmCen-/- immunization showed protection and an immune response comparable to leishmanization. LmCen-/- is safe since it was unable to cause disease even in immunocompromised mice, induces robust host protection against vector sand fly challenge and because it is marker free, can be advanced to human vaccine trials.

microbiology↗