以桿狀病毒表現系統生產H6亞型流感HA似病毒顆粒及其免疫效果之評估

Abstract

Vaccination for eliciting neutralization antibody against HA is the major prophylactic strategy to reduce diseases caused by an influenza virus infection. Virus-like particles (VLPs) are looking as a newly promised vaccine candidates due to their lack of viral genetic materials and morphological resemblance to authentic viruses. In this study, we want to develop a subunit vaccine against H6 subtype influenza viruses, which are frequently identified in wild birds or domestic poultry. For this purpose, HA gene from a local influenza strain, A/chicken/Taiwan/2838V/00 (H6N1), was constructed and expressed in Hi-5 insect cells. The results demonstrated that the expression of HA on cell membrane conferred the infected Hi-5 cells with the function to agglutinate erythrocytes. Furthermore, the hemagglutination was also detectable from the medium, which suggested some HA molecules were released into extracellular environment. The infection has been optimized to have a highest HA expression level using the multiplicity of infection (MOI) of 0.001. 20-60% sucrose density gradient ultracentrifugation analysis was demonstrated that these extracellular HA molecules were released in a particle form with the buoyant densities between 1.16-1.21 g/cm3. The existence of HA-VLPs with a size of 80-120nm and the prominent HA spikes were confirmed by electron microscopy. On the other hand, analysis of the HA protein in the intracellular, the hemagglutination was also detectable from the lysate, which suggested HA protein with function were produced in the intracellular. 20-60% sucrose density gradient ultracentrifugation analysis was demonstrated that these intracellular HA molecules were produced in a particle form with the buoyant densities between 1.13-1.17 g/cm3. The existence of HA-VLPs with a size of 80-100nm were confirmed by electron microscopy. The HA-VLPs have function from pH 6-10 down to 37℃. Animal immunization demonstrated that HA-VLPs elicit high titers of hemagglutination inhibition (HI) antibody to protect chickens. This study demonstrated that expression of influenza HA by baculovirus expression system is functional and can form HA-VLPs, and that can protect chicken.

預防流感病毒感染，主要以施打疫苗令接種個體產生對抗HA的中和性抗體為主，似病毒顆粒(virus-like particle) 被視為新一代的流感疫苗，乃因其不具病毒核酸且結構與真實病毒相似，具有安全性與效果佳的優點。本研究目的為利用桿狀病毒表現系統表現H6亞型流感病毒HA似病毒顆粒，並以此開發為防治家禽流行性感冒的次單位疫苗。實驗利用桿狀病毒表現系統在Hi-5昆蟲細胞表現H6N1亞型A/chicken/Taiwan/2838V/00病毒株的HA蛋白，經由血球吸附試驗證實受感染的Hi-5細胞會在細胞膜上表現具有功能的HA蛋白；而在血液凝集試驗發現在胞外培養基具有血球凝集的現象，顯示HA蛋白有被釋放到胞外；以不同病毒感染複數(MOI) 做HA蛋白表現最適化，發現在病毒感染劑量0.001時，HA蛋白會有最佳的表現量；將胞外培養基以20~60%蔗糖梯度離心，發現HA主要分布於浮力密度約為1.16~1.21 g/cm3，顯示HA蛋白可能形成顆粒結構。進一步利用穿透式電子顯微鏡觀察到大小約為80~120nm、帶有HA突刺狀的似病毒顆粒。本研究證實利用桿狀病毒表現系統表現H6亞型流感病毒HA蛋白，可形成帶有活性的似病毒顆粒。另一方面，進一步分析胞內HA蛋白，經由血液凝集試驗發現在胞內HA蛋白具有血球凝集的現象，顯示在胞內也可表現具有功能的HA蛋白；將胞內蛋白以20~60%蔗糖梯度離心，發現HA主要分布於浮力密度1.13~1.17 g/cm3，顯示HA蛋白同樣可能形成顆粒結構。利用穿透式電子顯微鏡觀察到大小約為80~100nm的似病毒顆粒。分析似病毒顆粒耐受性發現，當pH值為6~10、溫度低於37℃時，不影響其活性。動物免疫實驗證實雞隻免疫兩次添加佐劑的HA似病毒顆粒後，皆可產生具保護效果的血球凝集抑制 (HI)抗體。本研究證實利用桿狀病毒表現系統表現H6亞型流感病毒HA蛋白，可形成帶有活性且具保護效果的似病毒顆粒。