乙型肝炎人工纳米糖

StephenW

Artificial Nanodecoys For Hepatitis B
by Gang Liu
Credit: Wikimedia Commons

Chronic hepatitis B virus (HBV) infection places patients at high risk of death due to liver cirrhosis and hepatocellular carcinoma, and it remains a global public health issue. The receptors anchored on the cell membrane are generally key proteins for virus binding and entry, and they are also known as important therapeutic targets for various infectious diseases. Human sodium taurocholate co-transporting polypeptide (hNTCP) is a nine-transmembrane transporter consisting of 349 amino acids with a molecular mass of 56 kD. hNTCP has been demonstrated as a functional receptor of HBV and features essential high binding affinity for HBVpreS protein.


Cellular membrane vesicles (MVs) are directly formed from the natural cell membrane outward budding, they can reflect the antigenic content of the original cells, providing a new strategy for natural protein/vaccine delivery systems. We thus engineered MVs to display hNTCP receptors (hNTCP-MVs) and validated the antiviral effect of hNTCP-MVs to trick HBV both in vitro and in vivo. We confirmed that the nanomimics are able to recognize and bind to the dissociative HBV virion with high specificity and sensitivity. These findings highlight the novel roles of biomimetic binding strategies in which the unique capabilities of viral bio-interfacing are employed against HBV infection.

To obtain hNTCP-MVs, cell membrane derived from hNTCP-overexpressing cells was collected by a previously reported freeze-thaw process. After sonication and centrifugation, major hNTCP-MVs were harvested. A series of verifications demonstrate that hNTCP-MVs engineered with “mimics” of the multiple transmembrane HBV receptors are able to recognize and bind to the HBV virion with high specificity.

To investigate the potential of hNTCP-MVs blocking the binding and entry into host cells of the HBV virion, hNTCP-overexpressing cells (HBV infection cell model) were incubated with hNTCP-MVs at various concentrations immediately after HBV infection. We detected and analyzed HBV DNA and surface antigen (HBsAg) to measure the viral load in this study. In contrast to the controls, treatment with a 0.1 μg/μL dose of hNTCP-MVs significantly reduced HBsAg by 75.7 ± 2.5% (p < 0.001) and HBV DNA by 72.8 ± 1.6% (p < 0.001) in the supernatant 3 days post-infection (3 dpi). The HBV suppression effects of hNTCP-MVs were dose-dependent and were prolonged to 6 dpi.
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Next, we evaluated the antiviral effect of hNTCP-MVs in vivo in human liver chimeric mice (Hu-FRGS), a classic mouse model of HBV infection. In contrast to the Control-MVs treatment, the hNTCP-MVs treatment reduced serum HBsAg and HBV DNA levels by over 90% at 20 dpi. This indicated that hNTCP-MVs can effectively prevent HBV infection and the progression of viremia. Importantly, immunohistochemistry (IHC) staining showed that hNTCP-MVs treatment prevented the widespread expression of HBsAg and HBV core antigen (HBcAg) in chimeric human liver lobes. In addition, the treated mice showed normal physiological characteristics and liver function, suggesting the safety of the hNTCP-MVs regimen.

In summary, we successfully developed a new class of bioengineered nanodecoy anchoring natural HBV receptor (hNTCP) for use against HBV infection. The results show promise for inhibiting viral-host cell attack, suppressing viral load, and preventing viral spreading both in vitro and in vivo by mimicking a natural HBV infection process. Compared to soluble receptor proteins, MVs bestow natural properties of the source cell membrane in a straightforward manner, including unique virus bio-interfacing capabilities and lipid-based structures.

Furthermore, the approach avoids the potential risk of inhibitors and antagonists which might subvert the normal cellular functions of receptor proteins by inactivation of host receptors. Most importantly, to target different kinds of pathogens, the receptor can be changed, modified, and even further co-expressed by genetic engineering methods. Thus, the flexibility of bioinspired nanotechnology could potentially allow the prevention and treatment of multiple infectious diseases in future clinical translations.

These findings are described in the article entitled Bioinspired Artificial Nanodecoys for Hepatitis B Virus, recently published in the journal Angewandte Chemie. This work was conducted by Xuan Liu, Lunzhi Yuan, Liang Zhang, Yalin Mu, Xiaoling Li, Chao Liu, Peng Lv, Yali Zhang, Tong Cheng, Quan Yuan, Ningshao Xia, Xiaoyuan Chen, and Gang Liu from the Center for Molecular Imaging and Translational Medicine.

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Written By
Gang Liu

StephenW

乙型肝炎人工纳米糖
刘刚
图片来源：Wikimedia Commons

慢性乙型肝炎病毒（HBV）感染使患者因肝硬化和肝细胞癌而死亡的风险很高，并且仍然是全球公共卫生问题。锚定在细胞膜上的受体通常是病毒结合和进入的关键蛋白质，并且它们也被称为各种传染病的重要治疗靶标。人牛磺胆酸钠共转运多肽（hNTCP）是一种九跨膜转运蛋白，由349个氨基酸组成，分子量为56kD。 hNTCP已被证明是HBV的功能性受体，并且对HBVpreS蛋白具有必需的高结合亲和力。


细胞膜囊泡（MV）直接由天然细胞膜向外出芽形成，它们可以反映原始细胞的抗原含量，为天然蛋白质/疫苗递送系统提供新的策略。因此，我们设计MV以显示hNTCP受体（hNTCP-MV）并验证hNTCP-MV在体外和体内诱导HBV的抗病毒作用。我们证实纳米模拟物能够以高特异性和灵敏度识别并结合解离的HBV病毒粒子。这些发现强调了仿生结合策略的新作用，其中病毒生物接口的独特能力用于抗HBV感染。

为了获得hNTCP-MV，通过先前报道的冻融过程收集源自hNTCP过表达细胞的细胞膜。超声处理和离心后，收获主要的hNTCP-MV。一系列验证表明，用多种跨膜HBV受体的“模拟”工程改造的hNTCP-MV能够以高特异性识别并结合HBV病毒粒子。

为了研究阻断结合并进入HBV病毒粒子宿主细胞的hNTCP-MV的潜力，在HBV感染后立即将hNTCP过表达细胞（HBV感染细胞模型）与各种浓度的hNTCP-MV一起温育。我们检测并分析了HBV DNA和表面抗原（HBsAg），以测量本研究中的病毒载量。与对照组相比，用0.1μg/μL剂量的hNTCP-MVs治疗后3天内上清液中HBsAg显着降低75.7±2.5％（p <0.001），HBV DNA降低72.8±1.6％（p <0.001） - 感染（3 dpi）。 hNTCP-MV的HBV抑制作用是剂量依赖性的并且延长至6dpi。
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接下来，我们评估了hNTCP-MVs在人肝脏嵌合小鼠（Hu-FRGS）体内的抗病毒作用，这是一种典型的HBV感染小鼠模型。与Control-MVs治疗相比，hNTCP-MVs治疗在20 dpi时血清HBsAg和HBV DNA水平降低了90％以上。这表明hNTCP-MV可有效预防HBV感染和病毒血症的进展。重要的是，免疫组织化学（IHC）染色显示hNTCP-MVs治疗阻止了HBsAg和HBV核心抗原（HBcAg）在嵌合人肝叶中的广泛表达。此外，治疗小鼠显示出正常的生理特征和肝功能，表明hNTCP-MVs方案的安全性。

总之，我们成功开发了一类新的生物工程纳米结构锚定天然HBV受体（hNTCP）用于抗HBV感染。该结果显示出通过模拟天然HBV感染过程抑制病毒 - 宿主细胞攻击，抑制病毒载量和防止病毒在体外和体内扩散的希望。与可溶性受体蛋白相比，MV以直接的方式赋予源细胞膜的天然特性，包括独特的病毒生物界面能力和基于脂质的结构。

此外，该方法避免了抑制剂和拮抗剂的潜在风险，所述抑制剂和拮抗剂可能通过宿主受体的失活破坏受体蛋白的正常细胞功能。最重要的是，为了靶向不同种类的病原体，可以通过基因工程方法改变，修饰，甚至进一步共表达受体。因此，生物激发纳米技术的灵活性可能有助于在未来的临床翻译中预防和治疗多种传染病。

这些发现在最近发表于Angewandte Chemie期刊的题为Bioinspired Artificial Nanodecoys for Hepatitis B Virus的文章中有所描述。这项工作由刘璇，袁伦智，张亮，穆亚林，李晓玲，刘超，彭吕，张亚莉，佟成，全元，夏宁韶，陈晓媛，刘刚等分子影像中心主持和转化医学。

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