Role of the HTLV-1A and HTLV1-C inflammatory profile in disease

1.通过对160例HTLV-1a感染者的orf-I序列进行综合研究，以及对携带导致p8或p12优先表达的分子克隆的感染性进行动物试验，我们发现HTLV-1a在体内的有效持续和传播需要orf-I蛋白的联合作用。我们还发现，p8对于单核细胞的生产性感染是必不可少的，这两种蛋白的表达都使感染细胞完全抵抗MHC-I类限制的细胞毒性CD8杀伤。2.与细胞相关的HTLV-1至少可以通过三种不同的机制传播：病毒学突触、包括隧道纳米管在内的细胞管道和生物膜。然而，这些机制中的每一种对病毒传播的贡献仍然未知。我们已经证明HTLV-1p8通过增加细胞管道和隧道纳米管来增加病毒的传播。最近，我们发现核苷类似物阿糖胞苷(阿糖胞苷，AraC)抑制p8介导的TNT形成，使病毒传播减少30%，从而提供了一种部分抑制HTLV-1在体内传播的治疗方法。目前的研究和未来计划1.HTLV-1A和HTLV-1A/C病毒和orf-I cDNA的功能研究。我们已经合成了来自HTLV-1C的双重剪接的rex-orf-I mRNAs的cDNA，该mRNAs与orf-I并列在Rex的第一个外显子上，并证明它产生p16，一种增加自噬的蛋白质。我们用HTLV-1C的CLA-1-SAL-1片段与HTLV-1C的ORF-I和ORF-II的大部分片段互换，构建了一个嵌合病毒。由此产生的分子克隆是一种被命名为HTLV-1A/CDF的复制型病毒。我们计划通过取代完整的3‘端(HTLV-1A/C)并产生两种嵌合病毒的突变体来抑制rex-orf-I mRNA的剪接(HTLV-1A/CDFd16和HTLV-1A/CDFd16)作为对照，从而创建第二个HTLV-1A/C。这些病毒DNA克隆将被转导到支持HTLV-1复制的729 B细胞系中，并将用于感染原代CD4+T细胞。我们计划对带有慢病毒的单核细胞进行功能研究，慢病毒在原代单核细胞上表达p8或p16，并被HTLV-1AWT和orf-I敲除突变体、嵌合HTLV-1A/CDF WT或rex-orf-I敲除病毒(HTLV-1A/CDFd16)感染。我们将在体外评估炎性小体的激活、自噬以及原代单核细胞和T细胞产生的炎性细胞因子和趋化因子的水平和类型。2.人源化小鼠HTLV-1A和HTLV-1A/C的感染。我们有初步数据表明，我们的HTLV-1A分子克隆可以通过受感染的照射后的CD4+T细胞传播给人源化的NOD/SCID-YC-/-小鼠。我们观察到移植到人源化小鼠体内的人CD25+CD4+T细胞的增殖，导致这些CD4+T细胞广泛渗透到重要器官，如脾、高病毒负荷、体重减轻和死亡。为了探索HTLV-1在HU小鼠中的克隆性，我们应用了一种优化的高通量测序(HTS)方法来定位人类基因组中的病毒整合位点，并同时测量相应克隆的丰度。正如预期的那样，CD4+T细胞的增殖是多克隆的。我们预计HTLV-1a和HTLV-1a/C在小鼠身上引起这种增殖性疾病的能力不会有差异，因为这两种病毒的税收实际上是相同的。然而，这些研究将是证明嵌合HTLV-1A/C在体内具有病毒感染性的先决条件，然后它们才能用于非人类灵长类动物的研究。此外，这个小动物模型可能是测试阿糖胞苷在体内抑制病毒传播程度的基础。3.研究HTLV-1a和HTLV-1a/C感染猕猴和人的炎症过程。我们已经证明HTLV-1ORF-I基因敲除病毒和HTLV-1orf-I基因敲除病毒在体外感染单核细胞，在体内感染猕猴，但HTLV-1orf-I基因敲除病毒似乎没有持续存在。获得感染HTLV-1AWT或HTLV-1Aorf-I基因敲除病毒的PBMC，培养3d，检测贴壁细胞(巨噬细胞)产生细胞因子的能力。在两只动物的PBMC中病毒DNA阳性的时间点(8-10周)采集血液。我们发现，在感染HTLV-1Aorf-I基因敲除的动物中，IL-1B、IL-6和IL-8的水平高于HTLV-1AWT，这表明在体内，orf-I的缺失导致了本质上不同的炎症状态，体外也证明了这一点。我们计划通过用HTLV-1A感染4只猕猴和用HTLV-1ORF-I敲除病毒感染4只猕猴来扩展这项研究，以详细跟踪这两种病毒引起的炎症情况。我们还计划用HTLV-1a/CCS和HTLV-1a/CCSd16感染更多的猕猴，如果数据有保证的话。在所有动物研究中，我们将收集肺活检、支气管肺泡灌洗、血液、淋巴结、肠道活检和脊髓液，以量化病毒载量和全身炎症特征的差异。为了比较感染HTLV-1A和HTLV-1C的猕猴和人类的体外单核细胞的炎症特征，我们与护理HTLV-1C原住民的爱丽斯斯普林斯医院的澳大利亚医生以及东京的IMSUT医院和NIH的Steve Jacobson建立了合作伙伴关系，以直接从血浆或通过PBMC短期培养来研究他们的炎症特征。我们与澳大利亚研究人员的合作是一个大型的泛澳大利亚联盟，共享PBMC、培养方案、Luminex数据和用于比较人类和动物数据的试剂。

Accomplishments 1. By performing integrated studies on orf-I sequence in 160 HTLV-1A infected individuals and animal testing on the infectivity of molecular clones carrying polymorphism that result in preferential expression of p8 or p12, we established that efficient HTLV-1A persistence and spread in vivo requires the combined functions of the orf-I proteins. We also found that p8 is essential for productive infection of monocytes and that the expression of both proteins renders infected cells completely resistant to the MHC-class I restricted cytotoxic CD8 killing. 2. Cell-associated HTLV-1 can be transmitted by at least three different mechanisms: virological synapse, cellular conduits including Tunneling Nano Tubes, and biofilm. However, the contribution of each of these mechanisms to viral transmission remains unknown. We have demonstrated that the HTLV-1 p8 increases viral transmission by increasing both cellular conduits and Tunneling Nano Tubes. More recently, we found that inhibition of p8-medaited TNT formations by the nucleoside analog cytarabine (cytosine arabinoside, AraC) decreases viral transmission by 30%, thereby providing a treatment to partly curb the spread of HTLV-1 in vivo. Current Research & Future Plans 1. Functional studies with HTLV-1A and HTLV-1A/C viruses and orf-I cDNAs. We have synthesized the cDNAs derived from the doubly spliced rex-orf-I mRNAs of HTLV-1C that juxtapose the first exon of Rex in frame with orf-I and demonstrated that it produces p16, a protein that increases autophagy. We constructed a chimeric virus by swapping the Cla-1-Sal-1 fragment, which contains both the entire orf-I and most of the orf-II of HTLV-1A, with that of HTLV-1C. The resultant molecular clone is a replicating virus designated as HTLV-1A/CDF. We plan to create a second HTLV-1A/C by substituting the entire 3' end (HTLV-1A/C) and generating mutants of both chimeric viruses to inhibit the splicing of the rex-orf-I mRNA (HTLV-1A/CDFd16, and HTLV-1A/CDFd16,) as a control. The viral DNA clones will be transduced in the 729 B cell line that supports HTLV-1 replication and will be used to infect primary CD4+ T cells. We plan to perform functional studies on monocytes with lentiviruses that express p8 or p16 on primary monocytes and are infected by HTLV-1A WT and the orf-I knockout mutant, the chimeric HTLV-1A/CDF WT, or rex-orf-I knock-out virus (HTLV-1A/CDFd16). We will assess inflammasome activation, autophagy, and the level and type of inflammatory cytokines and chemokines produced by primary monocytes and T cells in vitro. 2. HTLV-1A and HTLV-1A/C infection of humanized mice. We have preliminary data that demonstrate that our HTLV-1A molecular clone can be transmitted to humanized NOD/SCID-yc-/- mice using infected, irradiated CD4+ T cells. We observed proliferation of human CD25+ CD4+ T cells engrafted in the humanized mice that causes extensive infiltration of these CD4+ T cells in vital organs such as the spleen, high viral burden, weight loss, and death. To explore HTLV-1 clonality in hu-Mice, we applied an optimized high-throughput sequencing (HTS) method to map viral integration sites in the human genome and simultaneously measure the abundance of the corresponding clones. The CD4+ T cell proliferation is polyclonal, as expected. We do not anticipate differences in the ability of HTLV-1A and HTLV-1A/C to cause this proliferative disease in mice, since the Tax is virtually identical in the two viruses. However, these studies will be prerequisite to demonstrate viral infectivity of the chimeric HTLV-1A/C in vivo before they can be used in studies in non-human primates. In addition, this small animal model may be foundational in testing the extent of inhibition of viral transmission by cytarabine in vivo. 3. Study the inflammatory profile of HTLV-1A and HTLV-1A/C infected macaques and humans. We have demonstrated that HTLV-1A WT and the HTLV-1 orf-I knockout viruses infect monocytes in vitro and macaquesin vivo, but the HTLV-1 orf-I knockout does not appear to persist. We obtained PBMCs infected with HTLV-1A WT or the HTLV-1A orf-I knockout virus, cultured them for 3 days, and measured the ability of adherent cells (macrophages) to produce cytokines. The blood was collected at a timepoint when both animals were positive for viral DNA in PBMCs (weeks 8-10). We found higher levels of IL-1B, IL-6, and IL-8 in the animal infected with the HTLV-1A orf-I knockout than that with the HTLV-1A WT, demonstrating that the absence of orf-I results in a qualitatively different inflammatory profile in vivo, as also demonstrated in vitro. We plan to extend this study by infecting 4 macaques with HTLV-1A and 4 with HTLV-1A orf-I knockout viruses to follow the inflammatory profiles caused by the two viruses in detail. We also plan to infect additional macaques with the HTLV-1A/CCS and the HTLV-1A/CCSd16 if warranted by the data. In all the animal studies, we will collect lung biopsies, bronchial alveolar lavage, blood, lymph nodes, gut biopsies, and spinal fluid to quantitate viral burden and differences in systemic inflammatory profiles. To compare the inflammatory profiles of ex vivo monocytes from macaques and humans infected by HTLV-1A and HTLV-1C, we have established collaborative efforts with Australian physicians at the Alice Springs Hospital that care for HTLV-1C infected Aborigines and with the IMSUT Hospital in Tokyo and Steve Jacobson at the NIH, who both care for HTLV-1A infected people, to study their inflammatory profiles directly in plasma or by short term cultures of PBMCs. Our collaboration with Australian researchers is a large pan-Australian consortium to share PBMCs, culture protocol, Luminex data, and reagents for the comparison of human and animal data.