Topical DNA immunization by modifying the hair follicle cycle

描述（由申请人提供）：皮肤充当病原体屏障，皮肤免疫系统装备良好，可产生有效的抗感染免疫应答。通过将DNA疫苗局部施用到皮肤上来诱导免疫应答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应答通常较弱，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以允许质粒编码的抗原基因表达。最近几项研究的数据指出，毛囊可能是局部应用DNA的入口。当将质粒DNA施加到皮肤上时，由质粒编码的基因的表达主要局限于毛囊中，并且仅偶尔在表皮的其他细胞中。此外，皮肤中正常毛囊的存在是局部DNA疫苗诱导特异性免疫应答所必需的，这一发现进一步表明毛囊是局部应用的质粒DNA的入口。正常的毛囊在生长期（生长期）和静止期之间循环。据报道，生长阶段的毛囊是开放的，皮肤表面的异物可以穿透，而静止阶段的毛囊是封闭的。事实上，当应用区域中的毛囊被诱导进入毛发生长期开始（早期生长）阶段时，局部应用于皮肤上的报告基因的表达显著增强。基于所有这些发现，我们假设当应用区域中的毛囊被诱导进入毛发生长期开始阶段时，由局部DNA疫苗诱导的免疫应答将增强。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，我们已经产生了一些有希望的数据，强烈支持这一假设，表明局部免疫通过应用DNA疫苗到皮肤区域的毛囊，或诱导到毛发生长初期的发病阶段代表一个可行的免疫方式。然而，在人类中验证这种方法之前，我们建议通过执行以下四个具体目标来进一步评估它：（i）阐明当施用区域中的毛囊被诱导进入毛发生长期（开始）阶段时，负责增强由局部DNA疫苗诱导的免疫应答的机制（目的#1），（ii）鉴定当施用区域中的毛囊处于或被诱导进入毛发生长期时影响由局部DNA疫苗诱导的免疫应答的因素（目标#2），（iii）评估使用这种方法的DNA疫苗诱导的免疫应答将保护动物免受致命病原体攻击的程度（目标3），并阐明局部应用于毛囊处于生长期阶段的皮肤区域的DNA疫苗的免疫诱导机制（目的#4）。我们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，以确保成功完成拟议的研究。这些结果将为我们在未来的试验中提高局部DNA疫苗的有效性奠定良好的科学基础。公共卫生相关性：拟议研究的成功完成将为通过修改毛囊周期在未来（临床）试验中开发和验证这种新型局部DNA免疫方法奠定良好的基础。这种有效的非侵入性局部DNA免疫模式预期适用于大规模免疫以预防人类和动物中的传染病。

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.