Ablation of HIV-1 infected cells by sustained bNAb expression from B-cells

项目2：摘要/摘要 针对HIV-1的广谱中和抗体(BNAbs)可以通过两种不同的机制抑制病毒复制。一个是中和病毒，防止感染。另一种是通过激活补体依赖性细胞毒(CDC)和抗体依赖性细胞毒(ADCC)来杀伤HIV-1感染细胞的能力。因此，bNAbs不仅有可能抑制新的病毒感染周期，而且还可以消除慢性感染细胞，这是治愈HIV-1疾病所必需的。然而，抗体的血清浓度必须在很长一段时间内保持在治疗水平才能清除HIV-1感染细胞，因为持续感染的细胞驻留在深层组织中。因此，需要多次注射抗体，这可能是昂贵和劳动密集型的。此外，给予重组bNAbs可提高宿主对bNAbs的免疫反应，导致bNAbs的中和和细胞毒活性丧失，半衰期缩短。基因治疗载体，特别是将转基因整合到宿主染色体中的慢病毒/肿瘤逆转录病毒载体，可以在体内长期产生bNAbs。但针对bNAbs的免疫反应仍可降低bNAbs的抗病毒效果，缩短bNAb的表达时间。B细胞在生理上表达由基因重组和突变产生的各种有价值的抗体区域。众所周知，B细胞可以诱导对有价值区域的耐受。B细胞的这种能力以前被用来诱导对自身抗原的耐受，用于自身免疫性疾病的治疗，以及凝血因子IX用于治疗血友病。 因此，用bNAb表达载体转导B细胞可能会产生长期的bNAbs，而不会引起对bNAbs的免疫反应。我们已经开发出慢病毒载体，可以在全身给药后特异性转导所需的靶细胞类型。通过该慢病毒载体对B细胞的特异性转导，我们将试图通过避免针对bNAbs的免疫反应来长时间表达bNAbs。我们还将尝试通过将转化的B细胞分化为长寿的浆细胞和记忆性B细胞来延长bNAb的表达持续时间。然后，我们将研究从B细胞表达的bNAbs是否可以消除小鼠体内感染HIV-1的细胞。由于常用的小鼠没有正常的补体，我们将使用一种新型的小鼠模型，该模型具有完整的补体系统，以充分发挥bNAb的CDC活性。接下来，我们将研究B细胞和T细胞表达的抗HIV-1转基因bNAbs是否能够协同消除B细胞中感染HIV-1的细胞。最后，我们将通过分析转导细胞的生物分布和细胞类型来研究系统地应用我们的慢病毒载体能否在非人类灵长类动物(NHP)中特异性地转导B细胞。我们还将研究在NHP中表达的bNAbs的药代动力学和免疫反应。这些实验旨在开发一种新的基于bNAb的基因治疗方法，该方法可适用于其他传染病。

Project 2: Summary/Abstract Broadly neutralizing antibodies (bNAbs) against HIV-1 can inhibit viral replication by two distinct mechanisms. One is the neutralization of virus, which prevents infection. The other is its ability to kill HIV-1 infected cells through activation of complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC). Thus, bNAbs have the potential to not only inhibit new viral infection cycles but also eliminate chronically infected cells, which are necessary for cure of the HIV-1 disease. However, the serum concentrations of the antibodies must be kept at therapeutic levels for long periods of time to eliminate HIV-1-infected cells, because persistently infected cells reside in the deep tissue. Therefore, multiple administrations of the antibodies are required, which could be expensive and labor-intensive. In addition, administration of recombinant bNAbs can raise host immune reactions to the bNAbs, leading to loss of neutralization and cytotoxicity activities of bNAbs and shortening their halflife. Gene therapy vectors, especially lentiviral/oncoretroviral vectors that integrate their transgene into host chromosomes, can produce bNAbs in vivo for long periods. But immune reactions against the bNAbs can still decrease the antiviral efficacy of bNAbs and shorten the duration of bNAb expression. B cells physiologically express wide varieties of valuable antibody regions generated by recombination and mutations in genes. B cells are known to induce tolerance to the valuable regions. This ability of B cells was previously used to induce tolerance to self-antigens for therapy of autoimmune diseases and coagulation factor IX for treatment of hemophilia. Therefore, transduction of B cells with bNAb expressing vectors is likely to generate long-term bNAbs without inducing immune reactions to the bNAbs. We have developed lentiviral vectors that can specifically transduce desired target cell types after systemic administration. By specific transduction of B cells by this lentiviral vector, we will attempt to express bNAbs for long periods of time by avoiding immune reactions against bNAbs. We will also attempt to prolong the duration of bNAb expression by differentiating transduced B-cells to long-lived plasma cells and memory B-cells, which have long life-span. We will then investigate if bNAbs expressed from B-cells can eliminate HIV-1-infected cells in mice. Since commonly used mice do not have normal complement, we will use a novel type of mouse model that has an intact complement system for full CDC activity of the bNAb. We will next investigate whether bNAbs expressed from B cells and T cells engineered to express anti-HIV-1 transgene can synergistically eliminate HIV-1 infected cells in the B cells. Lastly, we will investigate whether systemic administration of our B-cell targeting lentiviral vector can specifically transduce B cells in non-human primates (NHP) by analyzing biodistribution and cell types of transduced cells. We will also investigate the pharmacokinetics of and immune reactions to bNAbs expressed in NHP. These experiments are designed to develop a novel bNAb-based gene therapeutic approach, which can be applicable to other infectious diseases.