Department of Virology, Lomonosov Moscow State University, Moscow 119991, Russia.
Curr Pharm Des. 2013;19(31):5587-600. doi: 10.2174/13816128113199990337.
A new approach for super-expression of the influenza virus epitope M2e in plants has been developed on the basis of a recombinant Tobacco mosaic virus (TMV, strain U1) genome designed for Agrobacterium-mediated delivery into the plant cell nucleus. The TMV coat protein (CP) served as a carrier and three versions of the M2e sequence were inserted into the surface loop between amino acid residues 155 and 156. Cysteine residues in the heterologous peptide were thought likely to impede efficient assembly of chimeric particles. Therefore, viral vectors TMV-M2e-ala and TMV-M2e-ser were constructed in which cysteine codons 17 and 19 of the M2e epitope were substituted by codons for serine or alanine. Agroinfiltration experiments proved that the chimeric viruses were capable of systemically infecting Nicotiana benthamiana plants. Antisera raised against TMV-M2e-ala virions appear to contain far more antibodies specific to influenza virus M2e than those specific to TMV carrier particle (ratio 5:1). Immunogold electron microscopy showed that the 2-epitopes were uniformly distributed and tightly packed on the surface of the chimeric TMV virions. Apparently, the majority of the TMV CP-specific epitopes in the chimeric TMV-M2e particles are hidden from the immune system by the M2e epitopes exposed on the particle surface. The profile of IgG subclasses after immunization of mice with TMV-M2e-ser and TMV-M2e-ala was evaluated. Immunization with TMV-M2e-ala induced a significant difference between the levels of IgG1 and IgG2a (IgG1/IgG2a=3.2). Mice immunized with the chimeric viruses were resistant to five lethal doses (LD50) of the homologous influenza virus strain, A/PR/8/34 (H1N1) and TMV-M2e-ala also gave partial protection (5LD50, 70% of survival rate) against a heterologous strain influenza A/California/04/2009 (H1N1) (4 amino acid changes in M2e). These results indicate that a new generation candidate universal nanovaccine against influenza based on a recombinant TMV construct has been obtained.
已开发出一种新方法，可在设计用于农杆菌介导递送至植物细胞核的重组烟草花叶病毒（TMV，U1 株）基因组的基础上，在植物中超表达流感病毒表位 M2e。TMV 外壳蛋白（CP）作为载体，将三个版本的 M2e 序列插入到氨基酸残基 155 和 156 之间的表面环中。异源肽中的半胱氨酸残基可能会阻碍嵌合颗粒的有效组装。因此，构建了 TMV-M2e-ala 和 TMV-M2e-ser 两种病毒载体，其中 M2e 表位的第 17 和 19 位半胱氨酸密码子被丝氨酸或丙氨酸密码子取代。农杆菌浸润实验证明，嵌合病毒能够系统感染本氏烟植物。针对 TMV-M2e-ala 病毒粒子产生的抗血清似乎含有针对流感病毒 M2e 的抗体远远多于针对 TMV 载体粒子的抗体（比例为 5:1）。免疫金电子显微镜显示，两个表位均匀分布且紧密包装在嵌合 TMV 病毒粒子的表面上。显然，嵌合 TMV-M2e 粒子表面暴露的 M2e 表位掩盖了大多数 TMV CP 特异性表位，使其免受免疫系统的攻击。用 TMV-M2e-ser 和 TMV-M2e-ala 免疫小鼠后 IgG 亚类的分布情况进行了评估。用 TMV-M2e-ala 免疫小鼠可显著改变 IgG1 和 IgG2a 的水平（IgG1/IgG2a=3.2）。用嵌合病毒免疫的小鼠可抵抗同源流感病毒株 A/PR/8/34（H1N1）的五个致死剂量（LD50），并且 TMV-M2e-ala 还能对异源流感病毒株 A/California/04/2009（H1N1）（M2e 中有 4 个氨基酸变化）提供部分保护（5LD50，存活率为 70%）。这些结果表明，已获得一种基于重组 TMV 构建体的新型通用流感纳米疫苗候选物。
