Vector Biological Studies in Leishmaniasis

存在于利什曼原虫前鞭毛体表面的多态性脂磷酸聚糖（LPG）介导与白蛉载体中肠的结合，并解释了物种限制的载体能力。L.发现来自西非的主要菌株缺乏以β-连接的半乳糖终止的侧链，这解释了它们不能与帕帕塔斯中肠结合，以及它们依赖于杜博斯奇中肠进行自然传播。安湖来自西非的主要菌株用L.编码半乳糖基转移酶的主要基因。表达一个或两个β 1-3 Gal侧链残基的转基因寄生虫在帕帕塔斯中存活，但表达长链聚半乳糖的转染子则不能。筛选L.来自非洲、中东、阿富汗、南亚和俄罗斯南部的大量分离物的主要LPG结构显示，在L.主要流行区，表明适应中肠受体的多态性内P. papatasi物种复杂的表达。 一个主要的努力已被导向量化的自然传播剂量的利什曼原虫哺乳动物宿主在叮咬感染的白蛉，这是目前未知的任何天然白蛉载体。需要定量数据来确定设计旨在模拟自然传播的实验性针头感染时应使用的寄生虫剂量，并揭示可能影响临床结果的自然剂量范围。建立了一种基于实时PCR的方法，用于确定单个感染白蛉吸血期间传播给活宿主的利什曼原虫前鞭毛体的数量。正如预期的那样，携带较重感染的苍蝇在进食时往往会传播大量的寄生虫。感染良好的苍蝇从来没有进食完成，经常吃不到中肠一半的血餐。传播到老鼠可以检测到从苍蝇没有采取任何可检测的血餐。迄今为止记录的剂量范围是1000-5000个寄生虫，尽管在1000以下的宿主组织中寄生虫检测的灵敏度需要提高。 沙蝇叮咬传播首次被用于评估实验性疫苗对利什曼病的有效性。用活寄生虫或高效灭活疫苗ALM+CpG接种C57 BL/6小鼠，并暴露于感染的白蛉叮咬或针攻击。在活疫苗接种的小鼠中，在24小时内在白蛉叮咬部位发现了保护性抗原特异性产生IFN-γ的CD 4 + T细胞，并在72小时内观察到大量增加。相比之下，在ALM+CpG接种的小鼠中，产生IFN-γ的CD 4 + T细胞向白蛉叮咬部位的募集延迟至攻击后1周，并且它们的数量显著减少。攻击后四周，ALM+CpG疫苗接种显示出对针接种的强保护，但对感染的白蛉叮咬没有保护，而活疫苗接种对两者都有保护。白蛉攻击与嗜中性粒细胞和巨噬细胞早期募集到叮咬部位相关，这可能损害了ALM+CpG免疫小鼠中的免疫表达。相比之下，在活的接种疫苗的小鼠中，大量效应细胞快速募集到白蛉叮咬部位似乎克服了这些抑制条件。这些观察结果与疫苗设计和开发高度相关，因为它们确定了针对L.少校

The polymorphic lipophoshoglycan (LPG) present on the surface of Leishmania promastigotes mediates binding to the sand fly vector midgut, and accounts for species restricted vector competence. The LPGs of L. major strains from West Africa were found to lack side chains that terminate in beta-linked galactose, explaining their failure to bind to midguts of P. papatasi, and their reliance on P. duboscqi for natural transmission. An L. major strain from West Africa was transfected with a family of L. major genes that encode galactosyl transferases. Transgenic parasites expressing one or two beta 1-3 Gal side chain residues were rescued for survival in P. papatasi, though transfectants expressing long chain poly gals were not. Screening of L. major LPG structures from an extensive number of isolates from Africa, the Middle East, Afghanistan, South Asia and Southern Russia revealed a remarkable elongation in side chain length moving from West to East in the L. major endemic zones, suggesting adaption to midgut receptor polymorphisms expressed within the P. papatasi species complex. A major effort has been directed toward quantification of the natural transmission dose of Leishmania to mammalian host during the bite of an infected sand fly, which is currently unknown for any natural sand fly vector. The quantitative data are needed to define the dose of parasites that should be used when designing experimental needle infections intended to mimic natural transmission, and revealing natural dose ranges that may influence clinical outcome. A real-time PCR-based method was developed to determine the number of Leishmania promastigotes transmitted to a live host during blood feeding by a single infected sand flies. As expected, flies bearing heavier infections tended to transmit a larger number of parasites while feeding. Well-infected flies never fed to completion and frequently took a blood-meal that less than half filled the mid-gut. Transmission to mice could be detected from flies that had taken no detectable blood-meal at all. Dose ranges so far documented are 1000-5000 parasites, though the sensitivity of parasite detection in host tissue below 1000 needs to be improved. Transmission by sand fly bite is being used for the first time to evaluate the efficacy of experimental vaccines against leishmaniasis. C57BL/6 mice were vaccinated with live parasites or a highly effective killed vaccine, ALM+CpG, and exposed to infected sand fly bites or needle challenge. In live-vaccinated mice, protective antigen-specific IFN-gamma-producing CD4+ T cells were found at the site of sand fly bite within 24 hours and a substantial increase was observed by 72 hours. In contrast, recruitment of IFN-gamma-producing CD4+ T cells to the site of sand fly bite in ALM+CpG vaccinated mice was delayed until 1 week following challenge, and their numbers were dramatically reduced. Four weeks post-challenge, ALM+CpG vaccination showed strong protection against needle inoculation, but no protection against infected sand fly bite, whereas live-vaccination protected against both. Sand fly challenge was associated with early recruitment of neutrophils and macrophages to the site of bite that may have compromised the expression of immunity in the ALM+CpG immunized mice. By contrast, the rapid recruitment of large numbers of effector cells to the site of sand fly bite in the live vaccinated mice appeared to overcome these suppressive conditions. These observations are highly relevant to vaccine design and development as they identify critical correlates for vaccine efficacy against L. major.