Mardanova Eugenia S, Kotlyarov Roman Y, Kuprianov Victor V, Stepanova Liudmila A, Tsybalova Liudmila M, Lomonossoff George P, Ravin Nikolai V
a Institute of Bioengineering, Research Center of Biotechnology of the Russian Academy of Sciences , Moscow , Russia.
b Research Institute of Influenza, Russian Federation Ministry of Health , St. Petersburg , Russia.
Bioengineered. 2016;7(1):28-32. doi: 10.1080/21655979.2015.1126017. Epub 2015 Dec 28.
The ectodomain of the conserved influenza matrix protein M2 (M2e) is a promising target for the development of a universal influenza vaccines. Immunogenicity of M2e could be enhanced by its fusion to bacterial flagellin, the ligand for Toll-like receptor 5. Previously we reported the transient expression in plants of a recombinant protein Flg-4M comprising flagellin fused to 4 tandem copies of the M2e. The use of self-replicating recombinant vector based on the potato virus X allowed expression of Flg-4M in Nicotiana benthaminana leaves at a very high level, up to about 1 mg/g of fresh leaf tissue. Intranasal immunization of mice with Flg-4M induced M2e-specific serum antibodies and provided protection against lethal challenge with different strains of influenza A virus. Here we show that immunization with Flg-4M not only generates a strong immune response, but also redirects the response from the carrier flagellin toward the M2e epitopes. Significant IgG response to M2e was also developed in bronchoalveolar lavages of immunized mice. Protective activity of Flg-4M upon lethal influenza challenge correlated with a decrease of virus titers in lungs relative to the control. Overall these data show the potential for the development of a plant-produced M2e-flagellin universal influenza vaccine.
保守的流感病毒基质蛋白M2（M2e）的胞外结构域是开发通用流感疫苗的一个有前景的靶点。通过将M2e与细菌鞭毛蛋白（Toll样受体5的配体）融合，可以增强其免疫原性。此前我们报道了一种重组蛋白Flg-4M在植物中的瞬时表达，该蛋白由与4个串联拷贝的M2e融合的鞭毛蛋白组成。基于马铃薯X病毒的自我复制重组载体的使用，使得Flg-4M在本氏烟草叶片中能够以非常高的水平表达，高达约1毫克/克新鲜叶片组织。用Flg-4M对小鼠进行鼻内免疫可诱导产生M2e特异性血清抗体，并提供针对不同甲型流感病毒株致死性攻击的保护。在这里我们表明，用Flg-4M免疫不仅能产生强烈的免疫反应，还能将反应从载体鞭毛蛋白重定向到M2e表位。在免疫小鼠的支气管肺泡灌洗液中也产生了对M2e的显著IgG反应。相对于对照，Flg-4M在致死性流感攻击后的保护活性与肺中病毒滴度的降低相关。总体而言，这些数据显示了开发植物生产的M2e-鞭毛蛋白通用流感疫苗的潜力。
