NOVEL ANTIGEN DISPLAY SYSTEM TO PRESENT MULTIPLE ANTIGENS AS HIV VACCINES

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 E2DISP是嗜热脂肪杆菌丙酮酸脱氢酶亚基E2的工程化蛋白结构域，可用于表达异源多肽或蛋白质的N-端融合。E2DISP自组装成24 nm的病毒样颗粒(VLP)，显示多达60个外来抗原副本。其目的是：(1)设计具有保守中和决定簇的HIV-E2包膜(Env)融合蛋白，并确定它们在诱导中和抗体(NAB)方面的有效性；(2)探索免疫策略，以诱导持久的Th1 CD4、CTL记忆应答，同时保持持续的抗体应答；(3)探索E2DISPVLP与重组C3d佐剂的最佳呈现方式和联合给药；以及(4)优化在Prime-Boost方案中与其他有效增强细胞免疫和NAB应答的疫苗递送系统一起使用纯粹的和混合的E2-HIV VLP。在过去的一年里，我们开发了有效的方法来纯化和稳定纯颗粒和杂交颗粒，并表明用Gag-E2和Env-E2 VLP免疫的小鼠可以激发CD8+T细胞反应。在使用HIV Env融合蛋白60聚体颗粒在兔身上进行的实验中，我们成功地展示了在用DNA和E2蛋白VLP进行两次免疫后，仅在六周内就产生了高滴度的中和抗体。V3区域和gp41蛋白的部分片段都能在兔体内引发中和抗体，我们正在探索这种反应的广度。我们还在探索将这些融合蛋白通过微针递送到粘膜抗原提呈细胞。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. E2DISP, an engineered protein domain from Geobacillus stearothermophilus pyruvate dehydrogenase subunit E2, can be used to express N-terminal fusions of heterologous peptides or proteins. E2DISP self-assembles into 24 nm virus like particles (VLPs) displaying up to 60 copies of a foreign antigen. The aims are to: (1) Design HIV-E2 Envelope (Env) fusion proteins with conserved neutralization determinants and determine their effectiveness in eliciting neutralizing antibodies (NAbs); (2) Explore immunization strategies to elicit long lasting Th1 CD4, CTL memory responses while preserving sustained antibody responses; (3) Explore optimal presentation and co-administration of E2DISP VLPs with recombinant C3d-based adjuvants; and (4) Optimize the use of pure and hybrid E2-HIV VLPs in prime-boost regimens with other vaccine delivery systems effective in boosting cellular immunity and NAb responses. In the last year, we have developed effective methods for purifying and stabilizing pure and hybrid particles and have shown that mice immunized with the Gag-E2 and Env-E2 VLPs elicit CD8+ T cell responses. In experiments in rabbits using HIV Env fusion protein 60-mer particles, we have been successful in showing the development of high titers of neutralizing antibodies within only six weeks, after two immunizations with DNA and E2 protein VLPs. Both the V3 region as well as portions of the gp41 protein elicit neutralizing antibodies in rabbits, and we are exploring the breadth of this response. We are also exploring the delivery of these fusion proteins with microneedle delivery to the mucosal antigen-presenting cells.