A novel anti-HIV-1 bispecific bNAb-lectin fusion protein engineered in a plant-based transient expression system.
A novel anti-HIV-1 bispecific bNAb-lectin fusion protein engineered in a plant-based transient expression system.
复制标题
一种在植物瞬时表达系统中设计的新型抗 HIV-1 双特异性 bNAb-凝集素融合蛋白。
DOI：
10.1111/pbi.13090
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发表时间：
2019
影响因子：
13.8
通讯作者：
Matoba,Nobuyuki
中科院分区：
文献类型：
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作者：
SeberKasinger,LaurenE;Dent,MatthewW;Mahajan,Garima;Hamorsky,KrystalTeasley;Matoba,Nobuyuki
广泛中和抗体（bNAb）的发现是朝着更好地预防和治疗人类免疫缺陷病毒1型（HIV-1）迈出的重要一步。然而，有效的治疗可能需要抗HIV药物的组合，以避免病毒逃逸。这个问题的一个可能的解决方案是产生双特异性分子，其可以同时靶向两个脆弱位点，提供协同抑制作用。在这里，我们描述了一种新型双特异性融合蛋白的植物生产和抗HIV活性，该融合蛋白由CD 4结合位点特异性bNAb VRC 01的抗原结合片段（Fab）和抗病毒凝集素Avaren组成，其靶向HIV-1包膜的聚糖屏蔽（VRC 01 Fab ‐Avaren）。通过初步实验证明了VRC 01和Fc融合的Avaren二聚体（Avaren-Fc）的协同HIV-1中和活性，证明了该组合的合理性。使用GENEWARE®烟草花叶病毒载体，在本氏烟草中表达VRC 01 Fab ‐Avaren，并使用三步色谱法进行纯化。表面等离子体共振和ELISA证明Avaren和VRC 01 Fab部分均保留其各自的结合特异性。与VRC 01 Fab和Avaren的等摩尔组合相比，VRC 01 FaB-Avaren对来自A、B和C进化枝的代表性HIV-1毒株的中和活性增强。值得注意的是，VRC 01 Fab ‐Avaren的中和作用显著强于二价母体分子VRC 01 IgG和Avaren-Fc，IC 50值范围为48 - 310 pm。这些结果支持基于Avaren和bNAb的双特异性抗HIV蛋白的持续开发，其中基于植物的瞬时过表达系统将提供有效的蛋白质工程和生产平台。
The discovery of broadly neutralizing antibodies (bNAbs) has been a major step towards better prophylactic and therapeutic agents against human immunodeficiency virus type 1 (HIV‐1). However, effective therapy will likely require a combination of anti‐HIV agents to avoid viral evasion. One possible solution to this problem is the creation of bispecific molecules that can concurrently target two vulnerable sites providing synergistic inhibitory effects. Here, we describe the production in plants and anti‐HIV activity of a novel bispecific fusion protein consisting of the antigen‐binding fragment (Fab) of the CD4 binding site‐specific bNAb VRC01 and the antiviral lectin Avaren, which targets the glycan shield of the HIV‐1 envelope (VRC01Fab‐Avaren). This combination was justified by a preliminary experiment demonstrating the synergistic HIV‐1 neutralization activity of VRC01 and Fc‐fused Avaren dimer (Avaren‐Fc). Using the GENEWARE®tobacco mosaic virus vector, VRC01Fab‐Avaren was expressed inNicotiana benthamianaand purified using a three‐step chromatography procedure. Surface plasmon resonance and ELISA demonstrated that both the Avaren and VRC01Fabmoieties retain their individual binding specificities. VRC01Fab‐Avaren demonstrated enhanced neutralizing activity against representative HIV‐1 strains from A, B and C clades, compared to equimolar combinations of VRC01Faband Avaren. Notably, VRC01Fab‐Avaren showed significantly stronger neutralizing effects than the bivalent parent molecules VRC01 IgG and Avaren‐Fc, with IC50values ranging from 48 to 310 pm. These results support the continued development of bispecific anti‐HIV proteins based on Avaren and bNAbs, to which plant‐based transient overexpression systems will provide an efficient protein engineering and production platform.