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

描述(由申请人提供)：这项提案的总体目标是证明激光预照射可以促进子孢子体(SPZ)通过皮肤到肝脏的传递，从而增强基于SPZ的疫苗。预防疟疾感染的迫切需要仍然是一种有效的、经许可的抗疟疾疫苗。到目前为止，疟疾疫苗研发人员的“黄金标准”仍然是通过叮咬辐射(G)、SPZ感染的蚊子进行免疫。然而，将这一方法转化为临床面临着巨大的障碍，因为通过受感染的蚊子叮咬为大量人接种疫苗是不切实际的，而且这种免疫路线是不道德的。对于以SPZ为基础的疫苗，无论SPZ是否通过辐射或基因修饰而减弱，其效力都与SPZ进入肝脏的数量有关。虽然静脉注射(IV)可能是将SPZ输送到肝脏的最足够的方法，但它不是临床批准的免疫途径。另一方面，皮内接种(ID)不仅模拟自然感染，而且是一种临床可接受的途径，比静脉接种效率低得多，因为SPZ从皮肤向外迁移受到真皮内致密网络的限制，这可能对冷冻保存的gSPZ尤其如此，因为它们的运动性降低。我们发现，在注射前对接种部位进行激光预照射，可将gSPZ的皮肤到肝脏的流量增加到与静脉注射相当的水平。光照瞬间破坏了皮肤致密的微结构，允许SPZ相对自由地运动，并进入真皮的毛细血管和淋巴管。我们推测，这种激光治疗可以增加基于SPZ的疫苗的ID递送，当与用于SPZ诱导免疫的佐剂α-GalCer(a-半乳糖神经酰胺)结合时，可以进一步增强SPZ诱导的疟疾防御。为了验证这一假设，我们将验证激光介导的促进SPZ从皮肤到肝脏的传递是否 在激光照射部位ID注射不同数量的SPZ后40小时，通过量化肝脏中SPZ的数量，SPZ具有剂量和/或运动依赖性。这种剂量依赖效应随后将用新鲜分离或冷冻保存的gSPZ或遗传弱化的SPZ进行测试。我们还将在近交系和近交系小鼠模型中验证激光照射，然后对冷冻保存的gSPZ或基因减毒的SPZ进行ID接种，可以提供与静脉接种类似的对SPZ毒力感染的保护，以及激光和a-GalCer佐剂在保护免疫方面的协同效应。这项临床前研究如果成功，可能会使基于SPZ的疫苗离临床应用更近一步。 公共卫生相关性：将使用安全和非侵入性的激光进行短暂的皮肤照射，以促进减毒疟疾子孢子活疫苗经皮肤到肝脏的转运。将疫苗充分输送到肝脏将极大地提高疫苗的效力。

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.