Biology subjects

Hamano, S.

Publications and source records attributed to Hamano, S..

7 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

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↗

bioRxiv2022

Lysosomal vesiculophagy terminates STING signalling

Stimulator of interferon genes (STING) is essential for the type I interferon response against a variety of DNA pathogens1,2. Upon emergence of cytosolic DNA, STING translocates from the endoplasmic reticulum (ER) to the Golgi where STING activates the downstream kinase TBK1, then to lysosome through recycling endosomes (REs) for its degradation3-6. Although the molecular machinery of STING activation is extensively studied and defined7, the one underlying STING degradation has not yet been fully elucidated. Here we show an unanticipated mechanism termed "lysosomal vesiculophagy" dictates STING degradation. Airyscan super-resolution microscopy and correlative light/electron microscopy show that a cluster of STING-positive vesicles of an RE origin are directly encapsulated into lysosome. Screening of mammalian Vps genes, the yeast homologues of which regulate Golgi-to-vacuole transport8, shows that the endosomal sorting complexes required for transport (ESCRT) is essential for the STING encapsulation into lysosome. Knockdown of Tsg101 and Vps4, components of ESCRT9, results in the accumulation of STING vesicles in the cytosol, leading to the sustained type I interferon response. STING undergoes ubiquitination at REs and degradation of STING requires the ubiquitin-binding domain of Tsg101, ensuring the selective degradation of activated STING. Our results reveal a novel mode of autophagy that prevents hyperactivation of innate immune signalling and provide insights into the ER-Golgi-REs-lysosomes pathway for degradation of transmembrane proteins.

cell biology↗

bioRxiv2021

Skin resident memory cells generated by the L. major centrin gene deleted parasites mediate protective immune response analogous to leishmanization

Leishmaniasis is a vector-borne parasitic disease transmitted through the bite of a sand fly with no available vaccine for humans. Recently, we have developed a live attenuated Leishmania major centrin gene deleted parasite strain (LmCen-/-) that induced protection against a homologous and heterologous challenges. The protection is mediated by IFN{gamma} secreting CD4+ T effector cells and multifunctional T cells, which is analogous to leishmanization. Previously, skin tissue resident memory T cells (TRM cells) were shown to be crucial for host protection in a leishmanization model. In this study, we evaluated generation and function of skin TRM cells following immunization with LmCen-/- parasites and compared those with leishmanization. In the absence of recoverable LmCen-/- parasites, the skin of immunized mice showed functional TRM cells comparable to leishmanized mice. The generation of the skin TRM cells was supported by the induction of cytokines and chemokines essential for their production and survival. Following challenge infection with wild type L. major, TRM cells specific to L. major were rapidly recruited and proliferated at the site of infection in the immunized mice which was similar to leishmanization. Further, upon challenge, CD4+ TRM cells induced higher levels of IFN{gamma} and Granzyme B in the immunized and leishmanized mice than non-immunized mice. Taken together, our studies demonstrate that a genetically modified live attenuated Leishmania vaccine generates functional CD4+ TRM cells that mediate protection and can be a safer alternative to leishmanization.

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↗