Laser-facilitated delivery of malarial sporozoites from the skin to liver

描述（由申请方提供）：本提案的总体目标是证明激光预照射可以增强子孢子（SPZ）的皮肤至肝脏递送，从而增强基于SPZ的疫苗。一种有效的、获得许可的抗疟疾疫苗仍然是预防疟原虫感染的迫切需要。迄今为止，疟疾疫苗开发者的“黄金标准”仍然是通过感染SPZ的蚊子叮咬进行免疫接种。然而，将这种方法转化为临床面临着巨大的障碍，因为通过感染性蚊子的叮咬给大量人接种疫苗是不切实际的，而且这种免疫途径是不道德的。对于基于SPZ的疫苗，无论SPZ是通过辐照还是遗传修饰减毒，其效力都与运输到肝脏中的SPZ数量相关。虽然静脉注射可能最足以将SPZ递送至肝脏，但它不是临床批准的免疫途径。另一方面，皮内（ID）接种不仅模拟自然感染，而且也是临床上可接受的途径，其效率远低于IV，因为SPZ移出皮肤受到真皮中密集堆积网络的限制，这对于冷冻保存的gSPZ可能尤其如此，因为它们的运动性降低。我们发现，在ID之前对接种部位进行激光预照射将gSPZ的皮肤至肝脏运输增强至与IV注射相当的水平。光照短暂破坏了皮肤的致密微结构，允许SPZ相对自由地移动并进入真皮中的毛细血管和淋巴管。我们假设，这种激光治疗可以增强基于SPZ的疫苗的ID递送，并且当与α-GalCer（α-半乳糖神经酰胺）（SPZ诱导的免疫的佐剂）组合时，可以进一步增强SPZ诱导的针对疟疾的保护。为了验证这一假设，我们将验证激光介导的促进皮肤到肝脏的SPZ递送是否 SPZ剂量依赖性和/或运动性，通过将不同数量的SPZ ID注射到激光照射部位后40小时定量肝脏中的SPZ数量。然后将使用新鲜分离或冷冻保存的gSPZ或遗传减毒SPZ检测这种剂量依赖性效应。我们还将在远交系和近交系小鼠模型中验证激光照射后ID接种冷冻保存的gSPZ或遗传减毒的SPZ提供与IV接种类似的针对毒性SPZ感染的保护以及激光和α-GalCer佐剂对保护性免疫的协同效应。临床前研究如果成功，可能会使基于SPZ的疫苗更接近其临床应用。 公共卫生相关性：将采用安全和非侵入性激光进行短暂的皮肤照射，以促进减毒活疟疾子孢子疫苗的皮肤至肝脏运输。将疫苗充分输送到肝脏将大大提高疫苗的效力。

DESCRIPTION (provided by applicant): The overall objective of this proposal is to demonstrate that laser pre-illumination can enhance skin-to-liver delivery of sporozoites (SPZs), augmenting SPZ-based vaccines. An effective, licensed anti-malarial vaccine remains an urgent need for prevention against plasmodium infection. To date, the "gold standard" for malarial vaccine developers is still immunization by bites of irradiation (g), SPZ-infected mosquitoes. However, translation of this approach into the clinic faces formidable obstacles, due to impracticality of vaccinating a large number of people by bites of infectious mosquitoes and unethic of this route of immunization. For SPZ- based vaccines, irrespective of whether the SPZs are attenuated by irradiation or genetic modification, the efficacy is correlated with the number of SPZs trafficking into the liver. While intravenous (IV) injection may be most sufficient in delivering SPZs to the liver, it is not a clinically approved route of immunization. On the othe hand, intradermal (ID) vaccination, not only mimicking natural infection but also a clinically acceptable route, is much less efficient than IV because emigration of SPZs out of the skin is restricted by the densely packed network in the dermis, which may be particularly true for cryopreserved gSPZs owing to their reduced motility. We found that laser pre-illumination of the inoculation site prior to ID enhanced skin-to-liver traffic of gSPZs to a level comparable to IV injection. The illumination transiently disrupted the dense microarchitecture of the skin, permitting relatively free movement of SPZs and their entry into the capillaries and lymphatic vessels in the dermis. We hypothesize that this laser treatment can augment ID delivery of SPZ-based vaccines and when combined with a-GalCer (a-galactosylceramide), an adjuvant for SPZ-induced immunity, can further boost SPZ-induced protection against malaria. To test this hypothesis, we will verify whether laser-mediated facilitation of skin-to-liver delivery of SPZs is SPZ dose- and/or motility-dependent by quantification of the number of SPZs in the liver 40 hr after ID injection of varying numbers of SPZs into the site of laser illumination. This dose-dependent effect will be then tested with freshly isolated or cryopreserved gSPZs or genetically attenuated SPZs. We will also validate in outbred and inbred mouse models that laser illumination followed by ID inoculation of cryopreserved gSPZs or genetically attenuated SPZs affords similar protection against virulent SPZ infections as IV vaccination and a synergistic effect between laser and a-GalCer adjuvant on the protective immunity. The preclinical study, if successful, can potentially take SPZ-based vaccines one step closer to their clinical application. PUBLIC HEALTH RELEVANCE: Brief cutaneous illumination with a safe and noninvasive laser will be employed to facilitate skin-to-liver transportation of live attenuated malarial sporozoite vaccine. Sufficient delivery of the vaccine to the liver will greatly boost the efficacy of the vaccine.