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

项目2：摘要/摘要 针对HIV-1的广泛中和抗体（bNAb）可以通过两种不同的机制抑制病毒复制。一是中和病毒，防止感染。另一种是通过激活补体依赖性细胞毒性（CDC）和抗体依赖性细胞毒性（ADCC）杀死HIV-1感染细胞的能力。因此，bNAb不仅有可能抑制新的病毒感染周期，而且有可能消除慢性感染的细胞，这是治愈HIV-1疾病所必需的。然而，抗体的血清浓度必须长时间保持在治疗水平，以消除HIV-1感染的细胞，因为持续感染的细胞存在于深层组织中。因此，需要多次施用抗体，这可能是昂贵的和劳动密集型的。此外，施用重组bNAb可提高宿主对bNAb的免疫反应，导致bNAb的中和和细胞毒性活性丧失并缩短其半衰期。基因治疗载体，特别是将其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，可以在体内长时间产生bNAb。但针对bNAb的免疫反应仍会降低bNAb的抗病毒效力并缩短bNAb表达的持续时间。B细胞在生理上表达通过基因重组和突变产生的多种有价值的抗体区域。已知B细胞诱导对有价值区域的耐受性。B细胞的这种能力以前被用于诱导对自身抗原的耐受性以治疗自身免疫性疾病和诱导对凝血因子IX的耐受性以治疗血友病。 因此，用表达bNA B的载体转导B细胞可能产生长期bNAb而不诱导对bNAb的免疫反应。我们已经开发了慢病毒载体，可以在全身给药后特异性地抑制所需的靶细胞类型。通过这种慢病毒载体特异性转导B细胞，我们将尝试通过避免针对bNAb的免疫反应来长时间表达bNAb。我们还将尝试通过将转导的B细胞分化为寿命长的浆细胞和记忆B细胞来延长bNAb表达的持续时间。然后，我们将研究从B细胞表达的bNAbs是否可以消除小鼠中的HIV-1感染细胞。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，用于bNAb的完全CDC活性。我们接下来将研究从B细胞和被工程化以表达抗HIV-1转基因的T细胞表达的bNAb是否可以协同地消除B细胞中的HIV-1感染细胞。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我们的B细胞靶向慢病毒载体的全身给药是否可以特异性地抑制非人灵长类动物（NHP）中的B细胞。我们还将研究在NHP中表达的bNAb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感染性疾病。

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.