Structure and Immunogenicity of HIV-1 gp41 Membrane Prox

我们正在寻找三种不同的方法来诱导针对gp41MPR的中和抗体。其一是捕获并展示在固相蛋白脂质体表面的MPR微小蛋白的表达。第二种方法利用高免疫原性的乙肝表面抗原纳米微粒在选定的环境中展示MPR。我们正在寻求的第三种方法是与华盛顿大学的比尔·斯希德和大卫·贝克以及VRC的彼得·邝其志合作，稳定异源蛋白支架上的2F5表位。对于这种方法，扩展的环2F5表位精确地支架在蛋白质数据库中可用的结构定义的非HIV蛋白质上。一旦被移植，2F5表位-支架将被测试结合并结晶(由邝其志实验室)。该计划是生成三到四个支架，这些支架是通过结构建模产生的，将2F5与纳米分子亲和力结合在一起。一旦生产，2F5支架将在自由状态下结晶(没有2F5存在)。那些与2F5抗体诱导的构象非常接近的将接受免疫原性测试，以确定它们产生2F5表位特异性反应的能力，当然还将确定它们是否引发中和抗体。我们将单独或在Prime：Boost组合中测试2F5蛋白支架，以将免疫反应集中在独特且常见的2F5表位折叠上。2F5表位支架蛋白也将在Prime：Boost中与gp160pls结合或在乙肝颗粒环境中表达。对于任何MPR导向的方法，我们将使用评估是否需要异源T Help来增强免疫原性，或者Toll样受体(TLR)配基佐剂是否可以更好地激发针对潜在自身的抗体反应？类似于决定因素[13]。我们还计划在小鼠的自身免疫品系中测试一组选定的结构，以确定在这些动物中通常诱导的心磷脂样抗体是否可能是一种有益的反应，然后推动与MPR免疫原的亲和力成熟。 I.Minin Tang有几个项目正在进行中，这些项目与MPR相关，并与作为免疫原的环境PLs的改进和与PLs的Prime-Boost策略有关，并且已经使用几种表达系统来产生选定的结构核心，这是非常宝贵的。此外，她还做了一些细胞分析，这可能对解释对不同免疫原的免疫反应有一定价值。 A.免疫原性以确定我们是否可以利用膜诱导针对gp41MPR的抗体？各种问题正在测试中；MPR DNA免疫原性，MPR DNA+PADRE(异源T细胞帮助，因为我们不确定短MPR序列包含辅助表位)，MPR pls+PADRE？与EnvPL gp160的Prime-Boost结合。 用Barbara Capechi，用Barbara？S细菌表达系统启动Boost，在细菌的外膜小泡中出现大量的mpr重复序列。这是在兔子身上进行的，应该很快就会有一些结合和中和数据。 C.EnvPL的顺序增强，以驱动这些分支分离株(CD4BS，gp41MPR)之间通常保守的东西，而不是可变区定向反应。 D.我们已经进行了包括MPL或T辅助脂肽在内的研究，以增强其免疫原性。我们还改变了脂类(更类似于病毒膜的组成)来增强2F5/4E10的结合。 2.Sanjay Phogat正在与乙肝表面抗原合作，这种抗原形成22纳米颗粒，呈现MPR用于结合和免疫原性分析。杆状病毒表达和酵母表达产生高水平的纯颗粒。 哈维尔·格纳加正在检查由Ofek-Kuong结构定义的2F5表位？锁定？在选定的蛋白质支架上(与David Baker合作)进行生产、结合、免疫原性和结构检测。由于似乎有迹象表明，优质提振和半胱氨酸稳定确实有效，因此出现重大结果的可能性增加了。该项目未来的活动包括克隆、细菌和哺乳动物表达、复性、ELISAs/Biaccore、中和分析以及将结构信息整合到增强的免疫原设计中。

We are pursing 3 distinct means to elicit neutralizing antibodies directed against the gp41 MPR. One is the expression of MPR miniproteins captured and displayed on the surface of solid phase proteoliposomes. The second approach utilizes the highly immunogenic HepB surface antigen nanopaticles to display the MPR in selected contexts. The third approach we are pursuing is to stabilize the 2F5 epitope on heterologous protein scaffolds in collaboration with Bill Schief and David Baker at the University of Washington and Peter Kwong at the VRC. For this approach, the extended loop 2F5 epitope is precisely scaffolded onto structurally defined non-HIV proteins available in the protein data base. Once transplanted, the 2F5 epitope-scaffold will be tested for binding and crystallized (by the Kwong lab). The plan is to generate three to four scaffolds generated from the structural modeling that bind 2F5 with nanomolar affinity. Once produced, the 2F5 scaffolds will be crystallized in the free-state (without 2F5 present). Those that closely fit the 2F5 antibody-induced conformation will be tested for immunogenicity to determine their ability to generate 2F5 epitope specific responses and of course to determine if they elicit neutralizing antibodies. We will test the 2F5 protein scaffolds individually or in prime:boost combination to focus the immune response on the unique and common 2F5 epitope fold. The 2F5 epitope scaffold proteins will also be tested in prime:boost combination with the gp160 PLs or expressed in the HepB particulate context. For any of the MPR-directed approaches we will use assess if heterologous T help is required to enhance immunogenicity or if toll-like receptor (TLR) ligand adjuvants will better elicit antibody responses against the potentially ?self? resembling determinants [13]. We also plan to test a selected set of constructs in autoimmune strains of mice to determine if the cardiolipin-like antibodies commonly elicited in these animals might be a beneficial response to then drive to affinity maturation with the MPR immunogens. I. Min Tang has several projects ongoing that relate to the MPR and to improvements of the Env PLs as immunogens and prime-boost strategies with the PLs, and has been invaluable using several expression systems to generate selected cores for structure. Also she has done some cellular assays which may have some value in interpreting immune response to different immunogens. a. Immunogenicity to determine if we can elicit antibodies against the gp41 MPR employing membrane ? various issues are being tested; is the MPR DNA immunogenic, MPR DNA+PADRE (heterolgous T cell help since we are not sure that the short MPR sequence contains a helper epitope), MPR PLs +PADRE?in combination with prime-boost of EnvPL gp160. b. With Barbara Capechi, prime-boost with Barbara?s bacterial expression system where large numbers of MPR repeats are presented in the outer membrane vesicles of bacteria. This is in progress in rabbits, should have some binding and neutralization data soon. c. Sequential boosting of EnvPL in order to drive what is commonly conserved between these clade isolates (CD4BS, gp41 MPR) and not the variable region-directed response. d. We have conducted studies including MPL or T helper lipopeptides into the PLs to enhance their immunogenicity. We have also changed lipids (more similar to the viral membrane composition) to enhance 2F5/4E10 binding. II. Sanjay Phogat is working with the Hepatitis B surface antigen which form 22nM particles to present the MPR for binding and immunogenicity analysis. Production of high-levels of pure particles from baclovirus expression and yeast expression. III. Javier Guenaga is examining the 2F5 epitope as defined by the Ofek-Kwong structure ?locked? onto selected protein scaffolds (in collaboration with David Baker) to be examined by production, binding, immunogenicity and structure. Since there seems to be an indication that prime-boost, coupled with cysteine-stabilization actually works, the likelihood of significant outcome has increased. Future activities within this project include cloning, bacterial and mammalian expression, refolding, ELISAs/Biacore, neutralization assays and integrating structural information into enhanced immunogen design.