已经开发了构象稳定的Env三聚体作为用于诱导针对HIV-1的中和抗体的抗原。然而,这些可溶性抗原的非糖基化免疫显性碱基可能与中和抗体反应竞争。这促使尝试将Env三聚体偶联至有机或无机纳米颗粒,其中碱面向载体。这种定点耦合不仅可以封闭三聚体的碱基,而且通过重复显示增强B细胞活化。
■为了探索在微球上有序显示HIV-1Env对Env特异性B细胞活化的影响,我们使用了Bind&Bite,一种基于异二聚体卷曲螺旋肽的构象敏感抗原的新型共价偶联方法。通过在C端改造具有基本21-aa肽(肽K)延伸的三聚体HIV-1Env蛋白,我们能够使用带有反应位点的酸性互补肽(肽E)和生物素分子以定点方式共价生物素化抗原。这允许我们以定向方式将我们的抗原装载到链霉亲和素珠上。
■通过我们的Bind&Bite系统涂覆有HIV-1Env的微球显示i)通过构象抗HIVEnv广泛中和抗体(bNAb)增强的结合,ii)针对Env碱基的抗体的结合活性降低,iii)较高的Env特异性B细胞活化,和iv)与以无方向方向呈现HIV-1Env的珠子相比,调理后更有效地吸收。
■与通过Avi标签的定点生物素化相比,Bind&Bite,在替代共价蛋白质修饰方面提供了更大的灵活性,允许通过正交卷曲螺旋肽对选择性修饰多种蛋白质。因此,本研究中描述的通过肽K和肽E的结合和咬偶联方法为纳米颗粒疫苗设计提供了有价值的工具,其中需要正确折叠的抗原的表面缀合。
Conformationally stabilized Env trimers have been developed as antigens for the induction of neutralizing antibodies against HIV-1. However, the non-glycosylated immunodominant base of these soluble antigens may compete with the neutralizing antibody response. This has prompted attempts to couple Env trimers to organic or inorganic nanoparticles with the base facing towards the carrier. Such a site-directed coupling could not only occlude the base of the trimer, but also enhance B cell activation by repetitive display.
To explore the effect of an ordered display of HIV-1 Env on microspheres on the activation of Env-specific B cells we used Bind&Bite, a novel covalent coupling approach for conformationally sensitive antigens based on heterodimeric coiled-coil peptides. By engineering a trimeric HIV-1 Env protein with a basic 21-aa peptide (Peptide K) extension at the C-terminus, we were able to covalently biotinylate the antigen in a site-directed fashion using an acidic complementary peptide (Peptide E) bearing a reactive site and a biotin molecule. This allowed us to load our antigen onto streptavidin beads in an oriented manner.
Microspheres coated with HIV-1 Env through our Bind&Bite system showed i) enhanced binding by conformational anti-HIV Env broadly neutralizing antibodies (bNAbs), ii) reduced binding activity by antibodies directed towards the base of Env, iii) higher Env-specific B cell activation, and iv) were taken-up more efficiently after opsonization compared to beads presenting HIV-1 Env in an undirected orientation.
In comparison to site-directed biotinylation via the Avi-tag, Bind&Bite, offers greater flexibility with regard to alternative covalent protein modifications, allowing selective modification of multiple proteins via orthogonal coiled-coil peptide pairs. Thus, the Bind&Bite coupling approach via peptide K and peptide E described in this study offers a valuable tool for nanoparticle vaccine design where surface conjugation of correctly folded antigens is required.