用VLP表达HPIV3的HN和F蛋白及其介导的免疫应答对小鼠的保护作用研究

Human Parainfluenza virus type 3 (HPIV3) is a respiratory virus belongs to paramyxoviridae. It is second only to RSV as the main cause of acute lower respiratory tract diseases in infants and young children, as well as Immunocompromised individuals. There is currently no effective antiviral therapy or vaccines available. Virus-like particles (VLPs) are particles, composed of repeating structural proteins array on their surfaces and in their cores, and these structures mimic those of infectious viruses, but without the incorporation of viral genome. Thus, VLPs are incapable of the multiple rounds of infection typical of an infectious virus. However, native viral antigens arrayed on VLP surface can contribute to the potent immunogenicity of virus. .According to our previous research, HPIV3 matrix protein (M) is able to produce VLPs when expressed alone in eukaryotic cells, thus it is responsible for the assembly and release of the virus. Viral proteins including nucleoprotein (NP), phosphoprotein (P) and Hemagglutinin-neuraminidase protein (HN) could be incorporated into M-induced VLPs. Fusion protein (F) and HN are two main envelope glycoproteins and belong to core antigens of the virus. Therefore, it is hypothesized that VLPs contain F and HN will induce strong HPIV3-specific immune responses which may protect against HPIV3 infection. .In this study, based on our previous work on VLP, we try to combine the ability of M protein with the immunogenicity of F and HN to explore whether M-induced VLPs could effectively incorporate and express the epitope of F and HN on their surfaces. M，F and HN were co-expressed in eukaryotic cells , and high quality of M-F-HN VLPs were obtained via sucrose density gradient centrifugation, Furthermore, the immune responses and immunity induced by the VLP will be evaluated, with the purified F and HN protein as a comparison. By clarifying these questions, we are expected to provide new theoretical basis for the therapeutic approaches and vaccine developments of HPIV3.

人3型副流感病毒（HPIV3）是诱发婴幼儿急性下呼吸道疾病的主要病原之一，目前尚无预防HPIV3的有效疫苗。病毒样颗粒（VLPs）由病毒蛋白组成，在结构上模拟真病毒，不含基因组，无感染性。我们前期研究揭示HPIV3的基质蛋白M单独表达可形成VLP；病毒的NP、P和HN蛋白可被包装进M蛋白介导产生的VLP中。F和HN是HPIV3的两个包膜糖蛋白，属于病毒的核心抗原。本研究在前期对HPIV3 VLP的大量研究基础上，以M蛋白能包裹结构蛋白产生VLP为切入点，结合F和HN的免疫原性特点，初步阐明以M蛋白VLP为载体能更好地表达并展示F和HN的抗原表位。真核共表达M与F、HN蛋白，蔗糖密度梯度离心纯化M-F-HN VLP，探究此VLP在小鼠体内引发的免疫应答水平，与单独表达并纯化的F与HN蛋白的免疫效果相比较，评价VLP的免疫应答对小鼠的保护作用，以期为HPIV3的防治及疫苗研究提供新的理论依据。

人3型副流感病毒是在新生儿和婴儿中引起严重呼吸道疾病的主要病原体之一，可在小于6月龄婴儿中引发毛细支气管炎和肺炎，是仅次于RSV的另一大主要病原体。HPIV3可以在感染细胞中诱导形成包涵体（IBs）。IBs作为HPIV3的RNA复制转录的工厂，对子代病毒的形成起着重要作用。尽管一些研究已经对包涵体形成的相关蛋白进行了初步探索，但我们仍对HPIV3 IBs形成中涉及的宿主因子知之甚少。在此项目中，我们从病毒与细胞相互作用的角度出发，寻找参与包涵体形成的病毒蛋白和细宿主因子以及相互作用机制。通过免疫共沉淀和免疫荧光观察，我们发现细胞微丝调节蛋白cofilin与磷酸化蛋白P和核衣壳蛋白N形成的N-P复合体相互作用并定位到病毒包涵体中，从而帮助病毒的复制。当对cofilin进行RNA干扰时，病毒包涵体形成及病毒RNA的合成均明显下降。我们进一步发现HPIV3感染所诱导的cofilin磷酸化对其参与包涵体的形成过程十分重要。同时，通过构建cofilin截短突变体并进行免疫共沉淀分析，我们进一步确定了cofilin上与N相互作用的关键区域位于cofilin的C端。另一部分的研究我们聚焦于病毒核衣壳蛋白N与基质蛋白M之间相互作用。通过VLP出芽实验，免疫共沉淀及免疫荧光等方法确定了M蛋白中与N相互作用的功能区域位于M蛋白中143-182残基中。本课题的相关工作成果已发表在病毒学专业期刊上。希望我们的研究能够进一步了解人副流感病毒3型转录复制的具体机制及细节，为抗病毒药物和疫苗的研发提供新的思路和实验依据。