An Adjuvant-Independent Dual-Targeted (Multi-Function) Mucosal Vaccine Platform

 描述（由申请方提供）：大多数人类病原体通过粘膜途径进入。然而，获得许可的粘膜疫苗很少。因此，需要在粘膜部位刺激保护性免疫的新疫苗技术或佐剂。然而，目前的粘膜佐剂具有显著的局限性，包括毒性。我们建议开发/建立一种新的重组、无佐剂、粘膜疫苗平台。使用由靶向hFc γ RI的肺炎球菌表面蛋白A（PspA）抗原（Ag）组成的单[人Fc γ RI（hFc γ RI）]特异性原型融合蛋白（FP）证明了无佐剂粘膜疫苗平台的潜力。具体地，该原型FP增强免疫原性和针对粘膜S.当鼻内（i.n.）hFc γ RI转基因小鼠，在没有佐剂的情况下。我们现在提出创建一种双靶向（多功能）FP，其：1）通过FcRn介导的FP跨上皮转运，增强疫苗Ag向鼻相关淋巴组织（NALT）内表达hFc γ RI的Ag呈递细胞（APC）的递送。2)通过增加NALT内APC上的hFc γ RI交联，进一步增强Ag内化、树突状细胞（DC）成熟和Ag呈递/T细胞活化。重要的是，目前疫苗研究的重点主要是佐剂发现，最大限度地提高这种无佐剂疫苗平台的效力对于改变对佐剂需求的看法至关重要。为此，我们将：目的1）将FcRn靶向序列添加到含有FP的原型（单靶向）PspA中，以及将二价FP转化为三价FP。然后将在体外测试这种新型多功能FP依次增强Ag的跨上皮转运、Ag内化、DC成熟和Ag呈递/T细胞活化的能力。目的2：利用Sp PspA Ag粘膜疫苗模型和hFcRn/hFc γ RI表达小鼠优化FP平台免疫原性和保护效力。具体地，我们将比较二价与三价hFcRI靶向FP（加和减FcRn靶向组分）i.n.然后将选择最具保护性的FP进行进一步开发，并将检测针对多种Sp菌株与许可的Sp疫苗的免疫力和保护力。目标3：利用体外 图1示出了人鼻道的transwell模型，其由顶部孔中的人上皮细胞层（代表鼻腔）和下部孔中的人免疫细胞（代表NALT）组成。将检查优化的FP将Ag从上孔转运至下孔并随后增强下孔中的人T和B细胞应答的能力。将通过ELISA/ELISPOT测量Ag特异性抗体应答。将通过增加的CD25表达、T细胞增殖和细胞因子产生来测量T细胞活化。拟议的研究将是至关重要的，建立这种新的和创新的（双靶向/多功能）疫苗平台作为一个可行的无佐剂的粘膜免疫的方法。此外，它的成功将从根本上改变针对各种传染病病原体的疫苗生产/施用的模式。

 DESCRIPTION (provided by applicant): Most human pathogens enter via mucosal routes. Yet, there are very few licensed mucosal vaccines. Thus, there is a need for new vaccine technologies or adjuvants that stimulate protective immunity at mucosal sites. However, current mucosal adjuvants have significant limitations, including toxicity. We propose to develop/establish a novel recombinant, adjuvant-free, mucosal vaccine platform. The potential for an adjuvant-free mucosal vaccine platform is demonstrated using a mono [human FcγRI (hFcγRI)]-specific prototype fusion protein (FP) consisting of pneumococcal surface protein A (PspA) antigen (Ag) targeted to hFcγRI. Specifically, this prototype FP enhances immunogenicity and protection against a mucosal S. pneumoniae (Sp) challenge when administered intranasally (i.n.) to hFcγRI transgenic mice, in the absence of adjuvant. We now propose creating a dual-targeted (multi-functional) FP which: 1) Enhances delivery of vaccine Ag to hFcγRI-expressing Ag presenting cells (APCs) within the nasal-associated lymphoid tissue (NALT), via FcRn mediated transepithelial transport of FP. 2) Further enhances Ag internalization, dendritic cell (DC) maturation, and Ag presentation/T cell activation, via increased hFcγRI crosslinking on APCs within the NALT. Importantly, with the current focus of vaccine research primarily on adjuvant discovery, maximizing the potency of this adjuvant-free vaccine platform will be crucial to changing perceptions regarding the need for adjuvant. To achieve this, we will: Aim 1) Add an FcRn targeting sequence to the prototype (mono-targeted) PspA containing FP, as well as convert the bivalent FP to a trivalent FP. This novel multi-functional FP will then be tested in vitro for its ability to sequentially enhance transepithelial transport of Ag, Ag internalization, DC maturation, and Ag presentation/T cell activation. Aim 2: Optimize FP platform immunogenicity and protective efficacy utilizing the Sp PspA Ag mucosal vaccine model and hFcRn/hFcγRI-expressing mice. Specifically, we will compare bivalent vs. trivalent hFcRI-targeted FP (plus and minus the FcRn targeting component) i.n. The most protective FP will then be selected for further development and will also be tested for immunity and protection against multiple Sp strains versus a licensed Sp vaccine. Aim 3: Utilize an in vitro transwell model of the human nasal tract consisting of a human epithelial cell layer in the top well (representing the nasal lumen) and human immune cells in the lower well (representing the NALT). The ability of the optimized FP to transport Ag from the upper well to the lower well and subsequently enhance human T and B cell responses in the lower well will be examined. Ag-specific antibody responses will be measured by ELISA/ELISPOT. T cell activation will be measured via increased CD25 expression, T cell proliferation, and cytokine production. The proposed studies will be crucial to establishing this novel and innovative (dual-targeted/multi-functional) vaccine platform as a viable adjuvant-free approach for mucosal immunization. Furthermore, its success will fundamentally transform the paradigm for vaccine generation/administration against a wide array of infectious disease agents.