Topical DNA immunization by modifying the hair follicle cycle

描述（由申请人提供）：皮肤作为病原体屏障，皮肤免疫系统配备良好，可产生有效的抗感染免疫反应。通过在皮肤上局部应用DNA疫苗来诱导免疫反应是一种有吸引力的免疫方法。不幸的是，使用这种方法诱导的免疫反应通常很弱，这显然是由于大分子质粒DNA难以穿透皮肤角质层到达活的皮肤细胞，从而使质粒编码的抗原基因得以表达。最近的一些研究数据指出,毛囊可能的入口门户局部应用DNA。质粒DNA应用到皮肤时,基因的表达质粒编码的(s)在很大程度上局限于毛囊,只是偶尔在其他细胞在表皮。此外，外用DNA疫苗诱导特异性免疫应答需要皮肤中正常毛囊的存在，这一发现进一步表明，毛囊是外用质粒DNA的入口。正常的毛囊在生长期和休止期之间循环。据报道,毛囊处于增长阶段开放渗透外国对象在皮肤表面,而在休息阶段都关门了。事实上，当应用区域的毛囊被诱导进入生长初期时，局部应用于皮肤的报告基因的表达显著增强。基于这些发现,我们假设局部DNA疫苗诱导的免疫反应会增强毛囊时应用领域被诱导到生长期开始阶段。使用编码炭疽芽孢杆菌保护性抗原（PA）蛋白的质粒作为模型DNA疫苗，我们已经获得了一些有希望的数据，这些数据强烈支持这一假设，表明通过将DNA疫苗应用于毛囊处于或诱导进入毛原发病阶段的皮肤区域进行局部免疫是一种可行的免疫方式。然而,在人类验证这种方法之前,我们建议进一步评估它通过以下四个具体目标:(我)来阐明机制负责增强局部免疫反应引起的DNA疫苗毛囊时应用领域被诱导到生长期(发病)阶段(目标# 1),(2)确定因素影响局部DNA疫苗诱导的免疫反应,当毛囊在应用领域,或诱导,毛发生长初期阶段(目标# 2),(3)评估的程度对DNA疫苗诱导的免疫反应使用这种方法将保护动物免受致命的病原体挑战(目标# 3),并阐明DNA疫苗的免疫机制诱导局部使用在皮肤毛囊的地区在毛发生长初期阶段(目标# 4)。我们已经组建了一支在疫苗开发、经皮给药、皮肤生物学和组织病理学方面具有丰富经验的研究人员团队，以确保拟议研究的成功完成。结果奠定良好的科学基础我们改进在今后的试验中局部DNA疫苗的功效。公共卫生相关性：拟议研究的成功完成将为未来（临床）试验中通过改变毛囊周期来开发和验证这种新的局部DNA免疫方法奠定良好的基础。这种有效的非侵入性局部DNA免疫方式有望适用于大规模免疫，以预防人类和动物的传染病。

DESCRIPTION (provided by applicant): The skin acts as a pathogen barrier, and the skin immune system is well-equipped for the generation of effective anti-infection immune responses. The induction of immune responses by applying a DNA vaccine topically onto the skin is an attractive immunization approach. Unfortunately, the immune responses induced using this approach is generally weak, evidently due to the difficulty for the macromolecular plasmid DNA to penetrate through the skin stratum corneum to reach the viable skin cells to allow the antigen gene(s) encoded by the plasmid to be expressed. Data from several recent studies pointed out that the hair follicles are likely to be the portal of entrance for topically applied DNA. When plasmid DNA was applied onto the skin, the expression of the gene(s) encoded by the plasmid was largely confined in the hair follicles and only occasionally in other cells in the epidermis. Moreover, the finding that the presence of normal hair follicles in the skin was required for a topical DNA vaccine to induce specific immune responses further suggested the hair follicles as the portal of entrance for topically applied plasmid DNA. Normal hair follicles cycle between growth (anagen) and resting stages. It was reported that hair follicles in the growth stage are open for penetration by foreign objects on the skin surface, whereas those in the resting stage are closed. In fact, the expression of a reporter gene applied topically onto the skin was significantly enhanced when the hair follicles in the application area were induced into anagen onset (early growth) stage. Based on all these findings, we hypothesized that the immune responses induced by a topical DNA vaccine would be enhanced when the hair follicles in the application area were induced into anagen onset stage. Using a plasmid that encodes the protective antigen (PA) protein of Bacillus anthracis as a model DNA vaccine, we have generated some promising data that were strongly supportive of this hypothesis, indicating that topical immunization by applying a DNA vaccine onto a skin area where the hair follicles are in, or induced into, anagen onset stage represents a viable immunization modality. However, before validating this approach in humans, we propose to further evaluate it by carrying out the following four specific aims: (i) to elucidate the mechanisms responsible for the enhancement of the immune response induced by a topical DNA vaccine when the hair follicles in the application area are induced into anagen (onset) stage (aim # 1), (ii) to identify factors that affect the immune responses induced by a topical DNA vaccine when the hair follicles in the application area are in, or induced into, anagen stage (aim # 2), (iii) to evaluate the extent to which the immune responses induced by a DNA vaccine using this approach will protect the animals against a lethal pathogen challenge (aim # 3), and to elucidate the mechanisms of immune induction by a DNA vaccine applied topically onto a skin area where the hair follicles are in anagen stage (aim # 4). We have assembled a team of researchers with extensive experience in vaccine development, transdermal drug delivery, skin biology, and histopathology to safeguard the successful completion of the proposed studies. The outcomes will lay a sound scientific foundation for us to improve the efficacy of topical DNA vaccines in future trials. PUBLIC HEALTH RELEVANCE: The successful completion of the proposed studies will lay a sound foundation for the development and validation of this novel topical DNA immunization approach in future (clinical) trials by modifying the hair follicle cycle. Such an efficacious non-invasive topical DNA immunization modality is expected to be amenable for mass immunization to prevent infectious diseases in humans and animals.