能克服粘膜上皮细胞极性屏障的重组痘苗病毒HIV粘膜疫苗载体的构建

The mucosal surfaces are the main way of HIV entry and transmission. Thus a HIV vaccine being able to engender HIV-specific mucosal immune response as well as systemic immune response shall be more effective to prevent the invasion and transmission of HIV from the mucosal surfaces. A replicative vaccinia virus-based HIV vaccine is being evaluated in clinical trials by subcutaneous immunization. However, the inability of the current immunization route to induce mucosal immune response shall foresee its limited protection efficacy and the utility. With the financial support from the NSFC in the 2007-2009 period, we conducted research on the interaction of vaccinia virus HIV vaccine and mucosal epithelial cells and aimed to find out why the vaccinia virus HIV vaccine is not working well to induce mucosal immune response as a mucosal vaccine by mucosal immunization. We discovered that the polarity of epithelial cells is the critical factor for the inefficiency of the vaccinia virus-based HIV vaccine to induce mucosal immune response, in which the vaccine antigens are excluded to the basalateral secretion. Our further studies revealed that the loss of polarity enabled the expressed antigens to go to the basalateral side and thus increased the antigen intake and presentation by APC, which especially enhanced mucosal immune response besides systemic immune response. This work aims to develop a novel recombinant vaccinia virus vector that can overcome the polarity barrier of the epithelial cells and enhance the mucosal immune response of HIV vaccine. Toward achieving this goal, we propose to evaluate a number of novel vaccinia virus vector modifications by deletion of some genes that are hypothesized to be critical for the polarized distribution and release of virus and antigens. (1) We will test the hypothesis that vaccinia virus genes such as F13L gene which can interact with the host cell actin should be the genes for polarized distribution and release; (2) We will test the hypothesis that the polarized distribution and release of vaccinia virus and antigens can be changed through vector modification by the deletion of the polarity related genes; (3) We will test the hypothesis that the magnitude of immune response, especially the mucosal immune response can be significantly enhanced through vector modifications that specifically target the loss of polarity limitation. All in all, we propose to develop a novel recombinant vaccinia virus vector that can overcome the polarity barrier of the epithelial cells, which can be used for anti-HIV mucosal vaccine as well as for many other anti-viral mucosal vaccines.

HIV主要通过粘膜途径感染和传播，有效的HIV疫苗必须还能诱导HIV特异的粘膜免疫应答，从而有效地阻止病毒从粘膜途径的入侵、扩散和传播。基于复制型重组痘苗病毒载体的皮下接种HIV疫苗已经进入临床试验评估。但是，不能诱导粘膜免疫应答极大限制了这类候选疫苗的免疫保护效能和应用。在NSFC资助下，我们研究了痘苗病毒载体重组HIV疫苗粘膜免疫效应低下的原因，发现粘膜上皮细胞的极性阻碍了疫苗抗原向粘膜底侧面传输和递呈，并证明打破极化状态可促进疫苗抗原向上皮细胞底侧面的释放，提高抗原提呈细胞对抗原的摄取和提呈效率，显著增强免疫应答、尤其是粘膜免疫应答效应。本项目拟在前一课题研究基础上，筛选痘苗病毒载体极性分布和抗原释放相关基因，并进行修饰改造，构建能克服粘膜上皮细胞极性屏障的新载体，验证其免疫应答、尤其是粘膜免疫应答的相关性，获得一种新型的可用于包括HIV粘膜疫苗在内的基于重组痘苗病毒的粘膜疫苗载体。

复制型重组痘苗病毒载体在HIV疫苗中得到有效应用。提高该载体诱导针对疫苗抗原的免疫应答效应将提高该载体的应用价值。本项目选择痘苗病毒粒子装配转运和释放相关基因，对痘苗病毒A36R、F12L、A34R等基因进行了病毒的缺失或突变改造，构建获得了三个改造的痘苗病毒载体克隆rVTT△A36R-gfp、rVTT△F12L-gfp、rVTT-A34RK151E-gfp。在载体改造基础上构建获得了插入HIV-1外膜蛋白基因（Env）的重组痘苗病毒载体疫苗rVTT△A36R-Env及rVTT-Env-A34RK151E。实验证明，重组克隆rVTTΔA36R-Env感染黏膜上皮细胞释放病毒的能力明显降低，但不影响病毒向黏膜上皮细胞apical面的极性释放倾向。重组克隆rVTTΔA36R-Env通过鼻腔免疫小鼠不影响其在黏膜局部的病毒量，也不影响外源抗原的表达，但免疫应答明显减低。重组克隆rVTT-Env-A34RK151E同样不影响病毒向黏膜上皮细胞基底面的释放，滴鼻免疫Balb/c小鼠也不能提高免疫应答效应。然而，重组克隆rVTT-Env-A34RK151E感染Vero，BHK21以及小鼠肌肉细胞系C2C12明显提高病毒的释放，最终产生更多的子代病毒粒子；而且通过肌肉注射途径免疫小鼠，重组克隆rVTT-Env-A34RK151E能在小鼠肌肉组织中更有效地进行病毒复制与外源基因的表达，并诱导更强的免疫应答。细胞毒和对小鼠体重影响的测定显示重组克隆rVTT-Env-A34RK151E具有较好的安全性。因此，A34RK151E点突变是一种可行的提高痘苗病毒载体HIV-1疫苗免疫效应的载体改造方案。重组痘苗病毒载体克隆rVTT-Env-A34RK151E为进一步改进痘苗病毒载体HIV疫苗提供了一个新的范例和思路。

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