A Propagating Chimeric Particle as a Safe Alternative to Live-Attenuated Virus

描述(由申请人提供)：虽然高效抗逆转录病毒疗法(HAART)的使用通过控制病毒复制提高了艾滋病毒感染者的寿命和生活质量，但财务和后勤方面的缺陷以及临床副作用和耐药性的发展限制了其在全球的应用。因此，开发安全而有效的艾滋病毒疫苗仍然是全世界科学界和政界的长期目标。鉴于这一观察结果，本研究的总体目标是开发一种针对HIV的疫苗，该疫苗将减毒活病毒(LAV)优越且长寿的免疫力与非复制颗粒抗原的安全性相结合，以产生一种新型的、急性传播的嵌合粒子(PCP)疫苗。这些嵌合颗粒含有一个复制的VEE/SHV基因组，能够感染携带hCD4/hCCR5的敏感细胞，经历细胞质复制、基因组RNA封装、组装和颗粒释放。Vee-Shiv PCP不包含HIV ltrs或整合酶，因此增加了疫苗的总体安全性。由于这些颗粒的复制性，慢病毒蛋白将在接种的个体中表达一段较长的时间，从而诱导比非复制性抗原更全面的免疫反应。此外，嵌合颗粒具有固有的安全特性；VEE复制酶机制对干扰素敏感，因此限制了复制，直到对慢病毒蛋白的免疫反应清除病毒为止。与LAV相比，Vee-Shiv PCP疫苗的另一个优势是慢病毒结构蛋白与VEE的复制酶机制相结合，从而消除了野生型慢病毒在小鼠细胞中的复制限制。因此，与野生型慢病毒不同，Vee-Shiv PCP在存在或不存在颗粒成熟的情况下具有感染性，并且可以在小鼠细胞中无限制地高效复制。这项建议的主要目的是：1)用纯化的人胎肝造血干细胞(HuDKO)和表达Vee-Shiv PCP的DNA重组hCD4/hCCR5转基因(hCD4/R5TG)和BALB/c Rag2-/-c-/-小鼠，2)展示Vee-Shiv PCP的复制，3)评估安全性，以及4)评估PCP疫苗在hCD4/R5 TG和huDKO小鼠模型中的免疫原性。总体而言，在寻求针对HIV-1/AIDS的安全而有效的疫苗方面，传播嵌合粒子疫苗策略有望成为活疫苗的安全替代方案。公共卫生相关性：虽然由减毒活病毒诱导的免疫是HIV-1疫苗开发的黄金标准，但出于安全考虑，它们不能在人类身上使用。因此，Vee-Shiv传播嵌合粒子(PCP)疫苗被认为是一种安全的替代减毒活病毒的疫苗。这种原型嵌合颗粒疫苗包含慢病毒结构蛋白，可以诱导HIV特异性免疫，VEE的复制酶机制允许在BALB/c转基因hCD4/hCCR5和BALB/c Rag2-/-，c-/-小鼠中复制，这些小鼠是由来自人胎肝的人造血干细胞重组的，在本申请中描述的HIV感染的小鼠模型，以及人和非人灵长类动物中。

DESCRIPTION (provided by applicant): Although the use of highly active anti-retroviral therapy (HAART) has enhanced both the longevity and quality of life for HIV-infected individuals by controlling viral replication, financial and logistic drawbacks as well as clinical side effects and the development of drug resistance limits its worldwide use. Subsequently, the development of a safe yet effective vaccine for HIV remains the long term goal for both scientific and political communities worldwide. In light of this observation, the overall objective of this research is to develop a vaccine against HIV that combines the superior and long-lived immunity of a live-attenuated virus (LAV) with the safety of a nonreplicating particulate antigen to generate a novel, acutely-propagating chimeric particle (PCP) vaccine. These chimeric particles contain a replicating VEE/SHIV genome and are capable of infecting susceptible cells bearing hCD4/hCCR5, undergoing cytoplasmic replication, genomic RNA encapsidation, assembly, and particle release. The VEE-SHIV PCP does not contain the HIV LTRs or integrase therefore increasing the general safety of the vaccine. Due to the replicative nature of these particles, lentiviral proteins will be expressed for an extended period of time in vaccinated individuals thus inducing a more comprehensive immune response compared to nonreplicating antigens. Furthermore, the chimeric particle has an inherent safety feature; the VEE replicase machinery is sensitive to interferon thus limiting the replication until the immune response to the lentiviral proteins clears the virus. Another advantage of the VEE-SHIV PCP vaccine compared to LAV is that lentiviral structural proteins are combined with the replicase machinery from VEE, thus eliminating replication restrictions seen with wild-type lentiviruses in murine cells. Therefore, unlike wild-type lentiviruses, the VEE-SHIV PCP is infectious in the presence or absence of particle maturation and can efficiently replicate in murine cells without limitation. The main objectives of this proposal are to: 1) vaccinate hCD4/hCCR5 transgenic (hCD4/R5 Tg) and BALB/c Rag2-/-, c-/- mice reconstituted with human hematopoietic stem cells from human fetal liver (huDKO) with purified chimeric particles and DNA expressing the VEE-SHIV PCP, 2) demonstrate replication of the VEE-SHIV PCP, 3) assess safety, and 4) evaluate the immunogenicity of the PCP vaccine in hCD4/R5 Tg and huDKO mouse models. Overall, the propagating chimeric particle vaccine strategy is expected to be a safe alternative to live-attenuated vaccines in the quest for a safe, yet effective vaccine against HIV-1/AIDS. PUBLIC HEALTH RELEVANCE: Although the immunity induced by live-attenuated virus is the gold standard for HIV-1 vaccine development, safety concerns preclude their use in humans. Therefore, a VEE-SHIV propagating chimeric particle (PCP) vaccine is proposed as a safe alternative to live-attenuated virus. This prototype chimeric particle vaccine contains lentiviral structural proteins to elicit HIV-specific immunity, and the replicase machinery of VEE to permit replication in BALB/c transgenic hCD4/hCCR5 and BALB/c Rag2-/-, c-/- mice reconstituted with human hematopoietic stem cells from human fetal liver (huDKO) mouse models of HIV infection described in this application, as well as humans and non-human primates.