Innate & adaptive immunities in control of ocular HSV

描述（由申请方提供）：眼部原发性单纯疱疹病毒1型（HSV-1）感染后，病毒在眼部复制。眼部疾病可由HSV-1对角膜组织造成的直接损伤引起，或由感染后浸润角膜的细胞释放的细胞因子和趋化因子间接引起。病毒在眼部感染后的第7-10天被完全清除。免疫系统从眼睛中清除病毒所需的时间越长，随之而来的眼部疾病就越广泛和持久。减少眼睛中的病毒载量和加速病毒清除可能是减少潜伏期以及随后的复发性感染和视力丧失的最有效方法。因此，我们的主要目标是确定在减少眼睛中HSV-1复制、减少潜伏期的建立和从潜伏期再激活方面最有效的免疫应答。我们最近表明：（1）用5种HSV-1糖蛋白的混合物（5gP）免疫（gB，gC，gD，gE，gl）比用任何单一糖蛋白、gB+gD或活HSV-1疫苗免疫更有效;（2）5gP DNA免疫（对应于5gP蛋白的“裸”DNA）似乎甚至比5gP蛋白（亚基）免疫更有效，导致眼部病毒滴度进一步降低、病毒清除更快、三叉神经节（TG）潜伏期建立更短、IL-12和IFN-γ更高（附录1）。最近，我们已经表明，DNA免疫也刺激NK细胞，巨噬细胞，并产生显着更高的数量的CD 11 c+和CD 8 + T细胞的DNA免疫小鼠的角膜比蛋白免疫的小鼠。我们推测，DNA免疫后，通过树突状细胞（DC）的信号传导是重要的增强和维持的生产的CD 8 +/NK细胞在DNA免疫小鼠的角膜。我们进一步假设，在DNA免疫后，通过巨噬细胞的信号传导（IL-12产生）对于提供具有增强和维持IFN-γ产生所需的信号传导的CD 8 +/NK细胞是重要的。因此，为了进一步阐明负责减少眼部病毒复制、更快速清除HSV-1和减少用5gP DNA免疫的小鼠中的潜伏期的生物学和免疫学机制，我们提出实现以下特定目的： 1.证实了5gP DNA免疫的更高功效是由于 先天性和适应性免疫应答，而5gP蛋白（针对眼部HSV-1的最有效的蛋白疫苗）免疫的相对较低的功效主要是由于CD 4相关应答。 2.证实了使用IL-12和FLt 3L进一步刺激专职APC的假设， 佐剂将进一步增强CD 8 + T细胞和NK细胞的活性，导致DNA接种小鼠中眼部病毒复制、潜伏和再活化的更大减少。

DESCRIPTION (provided by applicant): Following primary herpes simplex virus type-1 (HSV-1) infection of the eye, virus replicates in the eye. Eye disease can result from direct damage inflicted on the corneal tissue by HSV-1, or indirectly, due to cytokines and chemokines released from cells infiltrating the cornea after infection. Virus is completely cleared between days 7-10 following ocular infection. The longer it takes for the immune system to clear the virus from the eye; the more extensive and protracted is the consequent ocular disease. Decreased virus load in the eye and accelerated virus clearance are probably the most efficient way to decrease latency and thus subsequent recurrent infections and loss of vision. Therefore, our main goal is to determine immune responses that are most efficacious at decreasing HSV-1 replication in the eye, reducing the establishment of latency, and reactivation from latency. We have shown recently that: (1) Immunization with a cocktail of 5 HSV-1 glycoproteins (5gP) (gB, gC, gD, gE, gl) is more efficacious than immunization with any single glycoprotein, gB+gD, or live HSV-1 vaccine; and (2) Immunization with 5gP DNA ("naked" DNA corresponding to the 5gP proteins) appeared even more efficacious than 5gP protein (subunit) immunization, resulting in further reduction of virus titers in the eye, more rapid viral clearance, lower establishment of latency in trigeminal ganglia (TGs), higher IL-12, and higher IFN-gamma (Appendix 1). Most recently, we have shown that DNA immunization also stimulates NK cells, macrophages and produces significantly higher numbers of CD11c+ and CD8 + T cells in the cornea of DNA-immunized mice than protein-immunized mice. We hypothesize that following DNA immunization signaling through dendritic cells (DCs) is important to the enhancement and maintenance of the production of CD8+/NK cells in the cornea of DNA-immunized mice. We further hypothesize that following DNA immunization signaling through macrophages (IL-12 production) is important in to the provision of the CD8+/NK cells with the signaling required to enhance and maintain IFN-gamma, production. Thus, to further elucidate the biologic and immunologic mechanisms responsible for reduced virus replication in the eye, more rapid clearance of HSV-1, and reduce latency in mice immunized with 5gP DNA, we propose to accomplish the following Specific Aims: 1. Confirm the hypothesis that the higher efficacy of 5gP DNA immunization is due to stimulation of both innate and adaptive immune responses, whereas the relatively lower efficacy of 5gP protein (the most effective protein vaccine against ocular HSV-1) immunization is mostly due to CD4-related responses. 2. Confirm the hypothesis that further stimulation of professional APCs using IL-12 and FLt3L as adjuvants will further enhance the activities of CD8 + T cells and NK cells leading to a greater reduction in ocular virus replication, latency, and reactivation in DNA-vaccinated mice.