Leishmaniasis is widely regarded as a vaccine-preventable disease, but the costs required to reach pivotal Phase 3 studies and uncertainty about which candidate vaccines should be progressed into human studies significantly limits progress in vaccine development for this neglected tropical disease. Controlled human infection models (CHIMs) provide a pathway for accelerating vaccine development and to more fully understand disease pathogenesis and correlates of protection. Here, we describe the isolation, characterization and GMP manufacture of a new clinical strain of Leishmania major. Two fresh strains of L. major from Israel were initially compared by genome sequencing, in vivo infectivity and drug sensitivity in mice, and development and transmission competence in sand flies, allowing one to be selected for GMP production. This study addresses a major roadblock in the development of vaccines for leishmaniasis, providing a key resource for CHIM studies of sand fly transmitted cutaneous leishmaniasis.
- MeSH
- fylogeneze MeSH
- hmyz - vektory parazitologie MeSH
- Leishmania major genetika růst a vývoj fyziologie MeSH
- leishmanióza kožní parazitologie přenos MeSH
- lidé MeSH
- modely nemocí na zvířatech MeSH
- myši inbrední BALB C MeSH
- paraziti genetika MeSH
- Psychodidae parazitologie MeSH
- sekvenování celého genomu MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Izrael MeSH
BACKGROUND: The peritrophic matrix (PM) is an acellular chitin-containing envelope which in most blood sucking insects encloses the ingested blood meal and protects the midgut epithelium. Type I PM present in sand flies and other blood sucking batch feeders is secreted around the meal by the entire midgut in response to feeding. Here we tested the hypothesis that in Sergentomyia schwetzi the PM creates a physical barrier that prevents escape of Leishmania parasites from the endoperitrophic space. METHODOLOGY/PRINCIPAL FINDINGS: Morphology and ultrastructure of the PM as well the production of endogenous chitinase in S. schwetzi were compared with three sand fly species, which are natural vectors of Leishmania. Long persistence of the PM in S. schwetzi was not accompanied by different morphology or decreased production of chitinase. To confirm the role of the PM in refractoriness of S. schwetzi to Leishmania parasites, culture supernatant from the fungus Beauveria bassiana containing chitinase was added to the infective bloodmeal to disintegrate the PM artificially. In females treated with B. bassiana culture supernatants the PM was weakened and permeable, lacking multilayered inner structure; Leishmania colonized the midgut and the stomodeal valve and produced metacyclic forms. In control females Leishmania infections were lost during defecation. CONCLUSIONS/SIGNIFICANCE: Persistence of the PM till defecation of the bloodmeal represents an important factor responsible for refractoriness of S. schwetzi to Leishmania development. Leishmania major as well as L. donovani promastigotes survived defecation and developed late-stage infections only in females with PM disintegrated artificially by B. bassiana culture supernatants containing exogenous chitinase.
- MeSH
- hmyz - vektory parazitologie fyziologie ultrastruktura MeSH
- králíci MeSH
- Leishmania major fyziologie MeSH
- Psychodidae parazitologie fyziologie ultrastruktura MeSH
- trávicí systém parazitologie ultrastruktura MeSH
- zvířata MeSH
- Check Tag
- králíci MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND Leishmania major is an Old World species causing cutaneous leishmaniasis and is transmitted by Phlebotomus papatasi and Phlebotomus duboscqi. In Brazil, two isolates from patients who never left the country were characterised as L. major-like (BH49 and BH121). Using molecular techniques, these isolates were indistinguishable from the L. major reference strain (FV1). OBJECTIVES We evaluated the lipophosphoglycans (LPGs) of the strains and their behaviour in Old and New World sand fly vectors. METHODS LPGs were purified, and repeat units were qualitatively evaluated by immunoblotting. Experimental in vivo infection with L. major-like strains was performed in Lutzomyia longipalpis (New World, permissive vector) and Ph. papatasi (Old World, restrictive or specific vector). FINDINGS The LPGs of both strains were devoid of arabinosylated side chains, whereas the LPG of strain BH49 was more galactosylated than that of strain BH121. All strains with different levels of galactosylation in their LPGs were able to infect both vectors, exhibiting colonisation of the stomodeal valve and metacyclogenesis. The BH121 strain (less galactosylated) exhibited lower infection intensity compared to BH49 and FV1 in both vectors. MAIN CONCLUSIONS Intraspecific variation in the LPG of L. major-like strains occur, and the different galactosylation levels affected interactions with the invertebrate host.
- MeSH
- druhová specificita MeSH
- galaktosa metabolismus MeSH
- glykosfingolipidy chemie metabolismus MeSH
- hmyz - vektory chemie fyziologie MeSH
- interakce hostitele a patogenu MeSH
- Leishmania major chemie fyziologie MeSH
- Phlebotomus parazitologie MeSH
- Psychodidae parazitologie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
BACKGROUND: Leishmania development in sand flies is confined to the alimentary tract and is closely connected with blood meal digestion. Previously, it has been published that activities of sand fly midgut proteases are harmful to Leishmania, especially to amastigote-promastigote transition forms. However, our experiments with various Leishmania-sand fly pairs gave quite opposite results. METHODS: We evaluated the effect of semi-digested midgut content on different life stages of Leishmania donovani and Leishmania major in vitro. Various morphological forms of parasites, including macrophage-derived amastigotes and transition forms, were incubated 2 h with midguts dissected at various intervals (6-72 h) post-blood meal or with commercially available proteinase, and their viability was determined using flow cytometry. In parallel, using amastigote-initiated experimental infections, we compared development of L. donovani in sand flies that are either susceptible (Phlebotomus argentipes and P. orientalis) or refractory (P. papatasi and Sergentomyia schwetzi) to this parasite. RESULTS: In vitro, sand fly midgut homogenates affected L. major and L. donovani in a similar way; in all sand fly species, the most significant mortality effect was observed by the end of the blood meal digestion process. Surprisingly, the most susceptible Leishmania stages were promastigotes, while mortality of transforming parasites and amastigotes was significantly lower. Parasites were also susceptible to killing by rabbit blood in combination with proteinase, but resistant to proteinase itself. In vivo, L. donovani developed late-stage infections in both natural vectors; in P. argentipes the development was much faster than in P. orientalis. On the other hand, in refractory species P. papatasi and S. schwetzi, promastigotes survived activity of digestive enzymes but were lost during defecation. CONCLUSIONS: We demonstrated that Leishmania transition forms are more resistant to the killing effect of semi-digested blood meal than 24 h-old promastigotes. Data suggest that Leishmania mortality is not caused directly by sand fly proteases, we assume that this mortality results from toxic products of blood meal digestion. Survival of L. donovani promastigotes in refractory sand flies until blood meal defecation, together with similar mortality of Leishmania parasites incubated in vitro with midgut homogenates of susceptible as well as refractory species, contradict the previously raised hypotheses about the role of midgut proteases in sand fly vector competence to Leishmania.
- MeSH
- gastrointestinální trakt enzymologie parazitologie MeSH
- králíci MeSH
- krev metabolismus MeSH
- Leishmania donovani fyziologie MeSH
- Leishmania major fyziologie MeSH
- Phlebotomus parazitologie MeSH
- proteasy metabolismus MeSH
- viabilita buněk MeSH
- zvířata MeSH
- Check Tag
- králíci MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Under laboratory conditions, hosts exposed twice to sand fly saliva are protected against severe leishmaniasis. However, people in endemic areas are exposed to the vector over a long term and may experience sand fly-free periods. Therefore, we exposed mice long- or short-term to Phlebotomus duboscqi bites, followed by Leishmania major infection either immediately or after a sand fly-free period. We showed that protection against leishmaniasis is limited to short-term exposure to sand flies immediately before infection. Our results may explain the persistence of leishmaniasis in endemic areas and should be taken into account when designing anti-Leishmania vaccines based on sand fly saliva.
- MeSH
- časové faktory MeSH
- hmyz - vektory imunologie parazitologie fyziologie MeSH
- kousnutí a bodnutí imunologie parazitologie MeSH
- Leishmania major fyziologie MeSH
- leishmanióza kožní imunologie parazitologie prevence a kontrola MeSH
- lidé MeSH
- myši inbrední BALB C MeSH
- myši MeSH
- Phlebotomus imunologie parazitologie fyziologie MeSH
- sliny imunologie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
