探討小鼠接種季節性流感病毒三醣化凝集素抗原免疫保護作用抵抗2009 H1N1新型流感病毒感染

Abstract

全球性的流感病毒（Influenza virus）傳染，主要源於人體免疫系統無法產生有效的抗體辨認流感病毒上的凝集素蛋白質（Hemagglutinin, HA），使得流感病毒在人跟人之間得以有效率的傳染開來。凝集素，是病毒表面上主要的醣蛋白（Glycoprotein），它具有專一性結合唾液酸（Sialic acid）的能力。流感病毒藉由凝集素結合人類呼吸道表皮細胞上的唾液酸，吸附和黏著住細胞並藉由細胞的吞噬作用（Endocytosis），進入宿主細胞（Host cell）。凝集素蛋白質被研究與發現是關鍵的抗原物質，如果將凝集素蛋白當成疫苗，可提供宿主細胞產生免疫反應，對抗病毒入侵人體。凝集素蛋白質的醣化對於蛋白質折疊以及受體的結合來說都十分重要，不過醣也可以遮蔽蛋白質上重要的、具有功能性的位置，讓宿主無法對病毒產生有效的抗體來對抗病毒的感染。之前的研究顯示黏著蛋白醣化位置具有三顆醣即可加速蛋白摺疊和增加穩定性，這意味著黏著蛋白上基本核心的三顆醣具有大體上穩定蛋白的功能，需再更進一步研究其它醣蛋白才能得知是否亦是如此。藉由這個研究我們獲得一個想法，或許凝集素蛋白醣化位置具有三顆醣是比其它醣化情形能成為更好的疫苗。在我們的研究，首先用酵素在酸鹼值4.9下將凝集素蛋白醣化位置上的多醣結構切成三醣，用去鹽管柱或離心過濾裝置移除酸溶液，用鎳樹酯純化三醣凝集素，之後用質譜等方法確定是否得到三醣凝集素，最後，探討三醣凝集素引發老鼠免疫反應的能力。實驗結果顯示八個凝集素蛋白醣化位置上的多醣結構大多切成三醣結構，三醣凝集素分子量也和預測之三醣凝集素分子量相似。此外，三醣凝集素二級結構與其它醣化型式凝集素相似。另外，在引發免疫反應的部分，三醣凝集素引發出的抗體效價並沒有比單醣凝集素引發出的抗體效價高，但比全醣凝集素高。這些研究結果或許對流感疾病的病理機轉有更進一步的瞭解與提供臨床治療有效疫苗的開發。

Influenza pandemics occur when influenza hemagglutinins (HA) are little recognized by immunity and the viruses transmit efficiently from human to human. HA is the major viral surface glycoprotein that binds to specific sialylated glycan receptors in the respiratory tract and allows the virus to enter the cell. It has been recognized as the key antigen in the host immune response to influenza virus in both natural infection and vaccination. The glycans of HA have been shown to be important for protein folding and receptor binding. Glycans can also mask important epitopes of protein so that the host may not produce effective antibodies to defeat the viruses. Previous study has showed that the core trisaccharide of an N-linked glycoprotein intrinsically accelerates folding and enhances stability. It means that the core triose of N-linked glycans has a general structure stabilizing effect. Here, we demonstrate that the core-trisaccharide HA (HAtg) could be a better protein vaccine than other HA glycoforms. Our analysis showed that the N-glycan of HAtg proteins were mostly two N-acetylglucosamines (GlcNAc) and one mannose (Man), and the molecular weight of HAtg was as expected. Furthermore, the secondary structure of HAtg is similar to HA with different glycosylations although it is in an aggregated form. The immunogenicity of HAtg was lower than the monoglycosylated HA (HAmg), but superior than the fullyglycosylated HA (HAfg). This study provides new insight into the vaccine design of HA glycoproteins against influenza.