Preclinical Studies of Vaccines for Pandemic H1N1 Influenza

需要流感的替代或辅助疗法，因为对目前使用的抗病毒药物的耐药性可能迅速出现。与CEL-SCI公司合作，我们在小鼠模型中测试了配体表位抗原呈递系统（LEAPS）技术作为流感病毒感染的新免疫治疗方法。流感-J-LEAPS肽是通过将来自人MHC I类分子的β 2 -微球蛋白链的结合配体（J-LEAPS）与来自流感病毒NP、M或HA蛋白的15至30个氨基酸长的肽缀合而合成的。用流感病毒-J-LEAPS肽（流感病毒-J-LEAPS）刺激DC并静脉内注射到感染的小鼠中。抗原特异性LEAPS刺激的DC有效减少流感病毒在肺中的复制并提高感染动物的存活率。此外，它们增强了肺部的流感特异性T细胞反应，并通过限制过度的细胞因子反应来降低疾病的严重程度，已知过度的细胞因子反应会导致流感病毒感染后的发病率和死亡率。我们的数据表明，流感病毒-J-LEAPS脉冲的DC减少病毒在肺中的复制，提高存活率，并调节保护性免疫应答，消除病毒，同时防止可能损伤宿主的过量细胞因子。这种方法显示出作为流感病毒感染的抗病毒治疗的辅助手段的前景。 HA是甲型流感病毒的主要病毒体表面蛋白，其快速抗原变异仍然是开发更广泛和更有效疫苗的主要挑战。然而，HA的一些区域，如接近病毒膜的茎区，在毒株之间和大多数亚型之间是高度保守的。疫苗设计中的一个基本问题是病毒表面上的HA茎区可接近广泛中和抗体的程度。与NCI的Sriram Subramaniams实验室合作，我们报告了在存在和不存在抗体C179的情况下，来自完整的2009年大流行H1N1病毒粒子上HA的冷冻电子断层扫描的3D结构，该抗体C179中和了表达广泛HA亚型的病毒，包括H1，H2，H5，H6和H9。通过拟合先前推导的三聚体HA的晶体结构的密度图，我们推导出的HA的分子表面参与与C179的相互作用的位置。使用计算方法来区分单个未配体的HA三聚体与那些结合C179抗体的HA三聚体，我们证明了病毒表面上75%的HA三聚体具有与茎结构域结合的C179。因此，尽管它们在病毒膜上紧密堆积，但完整病毒体上的大多数HA三聚体可用于结合靶向保守HA表位的抗干细胞抗体，从而建立了引发此类抗体的通用流感疫苗的可行性。

There is a need for alternative or adjunct therapies for influenza, because resistance to currently used antiviral drugs can emerge rapidly. In collaboration with CEL-SCI Corporation, we tested a ligand epitope antigen presentation system (LEAPS) technology as a new immune-based treatment for influenza virus infection in a mouse model. Influenza-J-LEAPS peptides were synthesized by conjugating the binding ligand derived from the β2 -microglobulin chain of the human MHC class I molecule (J-LEAPS) with 15 to 30 amino acidlong peptides derived from influenza virus NP, M, or HA proteins. DCs were stimulated with influenza-J-LEAPS peptides (influenza-J-LEAPS) and injected intravenously into infected mice. Antigen-specific LEAPS stimulated DCs were effective in reducing influenza virus replication in the lungs and enhancing survival of infected animals. Additionally, they augmented influenza-specific T cell responses in the lungs and reduced the severity of disease by limiting excessive cytokine responses, which are known to contribute to morbidity and mortality following influenza virus infection. Our data demonstrated that influenza-J-LEAPS pulsed DCs reduce virus replication in the lungs, enhance survival, and modulate the protective immune responses that eliminate the virus while preventing excessive cytokines that could injure the host. This approach shows promise as an adjunct to antiviral treatment of influenza virus infections. Rapid antigenic variation of HA, the major virion surface protein of influenza A virus, remains the principal challenge to the development of broader and more effective vaccines. Some regions of HA, such as the stem region proximal to the viral membrane, are nevertheless highly conserved across strains and among most subtypes. A fundamental question in vaccine design is the extent to which HA stem regions on the surface of the virus are accessible to broadly neutralizing antibodies. In collaboration with Sriram Subramaniams lab from NCI, we reported 3D structures derived from cryoelectron tomography of HA on intact 2009 pandemic H1N1 virions in the presence and absence of the antibody C179, which neutralizes viruses expressing a broad range of HA subtypes, including H1, H2, H5, H6, and H9. By fitting previously derived crystallographic structures of trimeric HA into the density maps, we deduced the locations of the molecular surfaces of HA involved in interaction with C179. Using computational methods to distinguish individual unliganded HA trimers from those that have bound C179 antibody, we demonstrated that ∼75% of HA trimers on the surface of the virus have C179 bound to the stem domain. Thus, despite their close packing on the viral membrane, the majority of HA trimers on intact virions are available to bind anti-stem antibodies that target conserved HA epitopes, establishing the feasibility of universal influenza vaccines that elicit such antibodies.