Viral transmission and pathogenesis in human tissues

人体组织中的病毒传播和发病机制

• 批准号: 10913219
• 负责人: Leonid B. Margolis
• 金额: $ 189.55万
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10913219
• 关键词: 2019-nCoV; ACE2; Acquired Immunodeficiency Syndrome; Affect; Alkanesulfonates; Antibodies; Arecaceae; Attention; B-Cell Activation; B-Lymphocytes; Binding; Biological Availability; Biological Markers; Blood Vessels; CCL2 gene; CCL23 gene; CCL3 gene; CD4 Positive T Lymphocytes; CD8-Positive T-Lymphocytes; CD80 gene; CD86 gene; CXCL10 gene; CXCL11 gene; CXCL13 gene; CXCL6 gene; CXCR3 gene; Cell Communication; Cell Culture Techniques; Cell Wall; Cell membrane; Cells; Cellulose; Characteristics; Charge; Clinical; Coagulation Process; Collaborations; Cross-Sectional Studies; Cytokine Activation; Data; Decision Trees; Derivation procedure; Development; Devices; Disease; Drug Delivery Systems; Drug Targeting; Early treatment; Effectiveness; Encapsulated; Endoglin; Enzyme-Linked Immunosorbent Assay; Event; Family; Fetal Death; Film; Filtration; Flow Cytometry; Functional disorder; Goals; HIV; HIV Infections; HIV-1; HLA-DR Antigens; Human; IL18 gene; IL3 Gene; Immunotherapy; Individual; Infection; Inflammatory; Interferon Type II; Label; Lead; Lesion; Link; Lymphocyte; Lymphoid Tissue; Macrophage Activation; Masks; Measures; Mediating; Membrane Proteins; Methods; Minor; Molecular; Multivesicular Body; Natural Compound; Natural Killer Cells; Nuts; Outcome; PGF gene; Pathogenesis; Pathology; Pathway interactions; Patients; Permeability; Persons; Phenotype; Plasma; Population; Pre-Eclampsia; Pregnancy Complications; Prevent viral transmission; Principal Component Analysis; Property; Proteins; Proteomics; Public Health; RANTES; Reporting; Research; Role; SARS-CoV-2 infection; SARS-CoV-2 inhibitor; SARS-CoV-2 spike protein; SARS-CoV-2 transmission; Sampling; South America; Structure of germinal center of lymph node; Subgroup; Sulfate; Surface; T memory cell; T-Lymphocyte; TFPI; TNF gene; TNFRSF5 gene; TNFSF10 gene; Techniques; Thinness; Time; Tissues; Transfection; Trees; Unexplained fetal death; Up-Regulation; Vesicle; Viral; Viral Load result; Viral Pathogenesis; Viremia; Virus; Virus Diseases; Virus Inhibitors; Woman; aerosolized; antimicrobial; antiretroviral therapy; biomarker identification; cell type; cohort; comorbidity; comparison control; cytokine; differential expression; experience; experimental study; extracellular vesicles; human tissue; immune activation; improved; in vivo; late endosome; men; monocyte; nano; nanoparticle; nervous system development; obstetric care; obstetrical syndromes; particle; prevent; programmed cell death ligand 1; random forest; receptor; respiratory; targeted treatment; therapeutic target; transmission blocking; transmission process; vesicular release; viral transmission

1. Cytokines in HIV disease We followed longitudinally 90 people living with HIV (PWH) (59 women and 31 men) already virally suppressed and on antiretroviral therapy (ART) on average for 6 years. We measured the concentrations of 41 cytokines in their blood plasma and followed their trajectory overtime. We found that long ART had a significant effect on the trajectory of 9 cytokines. In particular, the concentration of 8 cytokines continued to decrease over time (CD163; IP-10; CXCL13; I-TAC; MCP-1; MIG; MIP-3 and TNF-), while LBP concentration slightly increased over time. Our results suggest that long ART continues to modulate pro-inflammatory cytokines past the initial decrease in concentrations observed during ART suppressive therapy when viral load becomes undetectable. 2. Extracellular vesicle-associated cytokines in non-progressors among PWH We evaluated the cytokine profile associated with extracellular vesicles (EVs) in plasma samples from well-characterized cohorts of PWH with different virological control status. PWH were divided into 5 groups: ART-nave; ART-treated with undetectable viremia; elite controllers (EC) who controlled viremia (both transient controllers (TC) and persistent controllers (PC)); and uninfected individuals. Levels of 39 cytokines within and on the surface of EVs isolated from their stored plasma were quantified. Random forest, principal component analysis, and decision tree analyses were performed to identify specific cytokines as potential signatures of each study group. EC showed the highest levels of EV-associated cytokines, especially when PC were compared to TC. Higher levels of EV IL-18 were identified as a biomarker for EC population in the context of suppressed viremia, and higher levels of EV IL-3 and TRAIL were best at discriminating the PC phenotype from TC. In conclusion, higher levels of EV-associated cytokines in plasma may represent a new signature for the EC population. 3. EVs in immune activation of HIV-infected tissues under ART We investigated the mechanisms by which EVs from HIV-1-infected/ART-suppressed lymphoid tissues lead to immune activation. Using fluorescently labeled EVs and flow cytometry, we identified B cells as a prime target cell population of EVs from these tissues. Over time, the amount of pre-germinal center B cells increased followed by increased germinal center B cells, and B cell activation markers (CD40, CD80, CD86). By day 9, effector memory T cells increased, and natural killer cells increased activation markers (CD38, CD69, CD80, CD86, HLA-DR). The increase in cytokines was traced to multiple cell types including CD4 T cells (MIP-1a, MIP-1b, RANTES), CD8 T cells (IFN-g, MIP-1a, MIP-1b, RANTES, TNFa), B cells (MIP-1a, MIP-1b, RANTES), NK cells (RANTES) and monocytes (RANTES). Understanding the cascade of events triggered by EVs from may lead to potential therapeutic targets for suppression of improper immune activation in HIV-infected individuals under ART. 4. Localization of an HIV-1 protein on EVs We used several techniques to determine the localization of Nef in EVs. All approaches, including vesicle permeabilization, stripping of surface proteins, and remixing of EVs with Nef, led to the same conclusion: over 90% of the Nef molecules in the EV particles were located at the surface of the EVs. This result is consistent with Nef incorporation in the EVs via accession of the maturing late endosomes and/or the multivesicular bodies, and represent molecules associated with EVs during MVB fusion with the plasma membrane. Although only about 0.5% of total secreted Nef was associated with EVs, the Nef EVs were more biologically potent than soluble Nef. The distribution of Nef in EVs isolated from Nef-transfected cells was confirmed in EVs isolated from lymphocytes from PWH. Our study suggests that Nef can be released from cells in two forms: the dominant form is free protein, and the minor form is Nef associated with EVs with high bioactivity. Our results suggest that Nef EVs can be targeted by immunotherapy to treat HIV-associated co-morbidities. 5. EV-associated cytokines in complicated pregnancies leading to fetal death We found five proteins (CXCL6, endoglin, MIP-3, PlGF, and TFPI) to be different in concentrations in cases of fetal death complicated by preeclampsia compared to those without preeclampsia. Our data suggest that mechanisms of fetal death complicated with preeclampsia include the dysregulation of angiogenic, coagulation and inflammatory pathways. Among cases of fetal death, unsupervised clustering based on the proteins differentially expressed in either the EV or soluble fractions of patients with fetal death relative to controls revealed three major clusters of patients with distinct clinical and placental histopathological characteristics. Cluster 1 included 19 cases of fetal death that had 33 differentially expressed proteins in either the EV or the soluble fraction. Many of these cases of fetal death had two or more features consistent with maternal vascular malperfusion (MVM) and preeclampsia. Cluster 2 contained 12 cases of fetal death, and only PDL1 in the soluble fraction had different concentrations in this subgroup compared to controls. Fewer cases involved MVM lesions or preeclampsia but included the highest percentage of unexplained fetal death cases. Cluster 3 included 16 cases of fetal death with 21 proteins differentially expressed in either the EV or the soluble fraction of patients compared to the controls and fewer features of MVM and preeclampsia than cluster 1. Overall, our results demonstrate the value of including EV-associated biomarkers to improve the prediction of fetal death and understand the pathophysiology of this obstetrical syndrome. 6. Blocking SARS-CoV-2 transmission Nanocellulose particles were obtained from ivory nuts from a palm tree from the Arecaceae family that grows in tropical regions of South America and modified with dense sulfate charges. Due to its derivation from the endosperm of the palms seed, it was designated endospermic nanocellulose (ENC). The unique primary cell walls yielded more homogeneous and thinner cellulose nanoparticles compared to cell walls from other vegetal tissues. We evaluated the feasibility of sulfonated ENC as an inhibitor of viral transmission first by ELISA, which demonstrated effective blocking of SARS-CoV-2 S protein interaction with ACE2 in the presence of ENC, and allowed us to identify optimal working concentrations of ENC. We then used two different methods of blocking transmission of virus to cell cultures using ENC: a non-specific method of ENC encapsulation of SARS-CoV-2 pseudovirus particles or 293T-ACE2 target cells, and a highly specific method immobilizing molecular lures, anti-SARS-CoV-2 spike (S) protein antibodies and soluble ACE2 receptors, in ENC to bind virus. Both methods demonstrated that ENC effectively blocked cell infection by SARS-CoV-2 pseudoviruses, demonstrating the universal binding property of ENC and its usefulness for development of new strategies to prevent viral transmission. This material has potential applications as a film for masks and filters, to bind viral particles and increasing the effectiveness of these filtration devices, or to create adsorbent surfaces to capture or inactivate viruses. The concentrations of ENC used in these experiments will lend themselves easily to aerosolization making them ideal for such purposes. Nanoparticles that are deemed safe for in vivo use also have potential to enhance bioavailability of natural compounds, or for drug delivery.

1. HIV疾病中的细胞因子 我们对 90 名艾滋病毒感染者 (PWH)（59 名女性和 31 名男性）进行了纵向跟踪，这些人已经接受病毒抑制并接受抗逆转录病毒治疗 (ART)，平均时间为 6 年。我们测量了他们血浆中 41 种细胞因子的浓度，并随着时间的推移跟踪它们的轨迹。我们发现长期 ART 对 9 种细胞因子的轨迹有显着影响。特别是，8种细胞因子的浓度随着时间的推移持续下降（CD163；IP-10；CXCL13；I-TAC；MCP-1；MIG；MIP-3和TNF-），而LBP浓度随着时间的推移略有增加。我们的结果表明，当病毒载量变得不可检测时，长期 ART 会继续调节促炎细胞因子，超过 ART 抑制治疗期间观察到的最初浓度下降。 2. PWH 中非进展者的细胞外囊泡相关细胞因子 我们评估了具有不同病毒学控制状态的特征明确的感染者队列的血浆样本中与细胞外囊泡（EV）相关的细胞因子谱。 PWH 分为 5 组：ART-nave；经 ART 治疗后病毒血症未检出；控制病毒血症的精英控制者（EC）（瞬时控制者（TC）和持久控制者（PC））；以及未感染的个体。对从储存的血浆中分离出的 EV 内部和表面的 39 种细胞因子的水平进行了定量。进行随机森林、主成分分析和决策树分析，以确定特定细胞因子作为每个研究组的潜在特征。 EC 显示出最高水平的 EV 相关细胞因子，特别是当将 PC 与 TC 进行比较时。在病毒血症受到抑制的情况下，较高水平的 EV IL-18 被确定为 EC 群体的生物标志物，较高水平的 EV IL-3 和 TRAIL 最能区分 PC 表型和 TC。总之，血浆中较高水平的 EV 相关细胞因子可能代表 EC 人群的新特征。 3. EVs在ART下HIV感染组织免疫激活中的作用 我们研究了来自 HIV-1 感染/ART 抑制的淋巴组织的 EV 导致免疫激活的机制。使用荧光标记的 EV 和流式细胞术，我们确定 B 细胞是这些组织中 EV 的主要靶细胞群。随着时间的推移，前生发中心 B 细胞的数量增加，随后生发中心 B 细胞和 B 细胞激活标记物（CD40、CD80、CD86）也增加。到第 9 天，效应记忆 T 细胞增加，自然杀伤细胞增加激活标记（CD38、CD69、CD80、CD86、HLA-DR）。细胞因子的增加可追溯到多种细胞类型，包括 CD4 T 细胞（MIP-1a、MIP-1b、RANTES）、CD8 T 细胞（IFN-g、MIP-1a、MIP-1b、RANTES、TNFa）、B 细胞（ MIP-1a、MIP-1b、RANTES)、NK 细胞 (RANTES) 和单核细胞 (RANTES)。了解 EV 引发的级联事件可能会产生潜在的治疗靶点，用于抑制 ART 下 HIV 感染者的不当免疫激活。 4. HIV-1 蛋白在 EV 上的定位 我们使用了多种技术来确定 Nef 在电动汽车中的定位。所有方法，包括囊泡透化、表面蛋白剥离以及将 EV 与 Nef 重新混合，都得出相同的结论：EV 颗粒中超过 90% 的 Nef 分子位于 EV 表面。这一结果与 Nef 通过加入成熟的晚期内体和/或多泡体而掺入 EV 一致，并且代表了 MVB 与质膜融合期间与 EV 相关的分子。尽管只有约 0.5% 的分泌性 Nef 与 EV 相关，但 Nef EV 比可溶性 Nef 具有更强的生物学效力。 Nef 在从 Nef 转染细胞分离的 EV 中的分布在从 PWH 淋巴细胞分离的 EV 中得到了证实。我们的研究表明，Nef 可以两种形式从细胞中释放：主要形式是游离蛋白，次要形式是与具有高生物活性的 EV 相关的 Nef。我们的结果表明，Nef EV 可以通过免疫疗法来治疗 HIV 相关的并发症。 5. 复杂妊娠中EV相关细胞因子导致胎儿死亡 我们发现，与没有先兆子痫的胎儿相比，先兆子痫并发胎儿死亡的情况下，五种蛋白质（CXCL6、内皮糖蛋白、MIP-3、PlGF 和 TFPI）的浓度不同。我们的数据表明，子痫前期并发胎儿死亡的机制包括血管生成、凝血和炎症途径的失调。 在胎儿死亡病例中，基于胎儿死亡患者的 EV 或可溶性部分相对于对照差异表达的蛋白质进行无监督聚类，揭示了具有不同临床和胎盘组织病理学特征的三个主要患者群。第 1 组包括 19 例胎儿死亡病例，其 EV 或可溶性部分中有 33 种差异表达的蛋白质。许多胎儿死亡病例具有与母体血管灌注不良（MVM）和先兆子痫一致的两个或多个特征。第 2 组包含 12 例胎儿死亡病例，与对照组相比，该亚组中仅可溶性部分中的 PDL1 浓度不同。涉及 MVM 病变或先兆子痫的病例较少，但不明原因胎儿死亡病例的比例最高。第 3 组包括 16 例胎儿死亡病例，与对照组相比，患者的 EV 或可溶性部分中有 21 种蛋白质差异表达，并且 MVM 和先兆子痫的特征比第 1 组少。总体而言，我们的结果证明了将 EV 相关的纳入的价值生物标志物可以提高胎儿死亡的预测并了解这种产科综合征的病理生理学。 6. 阻断 SARS-CoV-2 传播 纳米纤维素颗粒是从生长在南美洲热带地区的槟榔科棕榈树的象牙坚果中获得的，并用浓密的硫酸盐电荷进行了改性。由于其源自棕榈种子的胚乳，因此被命名为胚乳纳米纤维素（ENC）。与其他植物组织的细胞壁相比，独特的初生细胞壁产生更均匀和更薄的纤维素纳米颗粒。我们首先通过 ELISA 评估了磺化 ENC 作为病毒传播抑制剂的可行性，结果表明，在 ENC 存在的情况下，可以有效阻断 SARS-CoV-2 S 蛋白与 ACE2 的相互作用，并使我们能够确定 ENC 的最佳工作浓度。然后，我们使用两种不同的方法使用 ENC 来阻止病毒向细胞培养物的传播：一种是 ENC 封装 SARS-CoV-2 假病毒颗粒或 293T-ACE2 靶细胞的非特异性方法，另一种是高度特异性的固定分子诱饵、抗病毒药物的方法。 -SARS-CoV-2刺突（S）蛋白抗体和可溶性ACE2受体，在ENC中结合病毒。两种方法都表明 ENC 有效阻止了 SARS-CoV-2 假病毒的细胞感染，证明了 ENC 的通用结合特性及其对于开发防止病毒传播的新策略的有用性。这种材料具有作为口罩和过滤器薄膜的潜在应用，可以结合病毒颗粒并提高这些过滤装置的有效性，或者形成吸附剂表面以捕获或灭活病毒。这些实验中使用的 ENC 浓度很容易雾化，使其成为此类目的的理想选择。被认为可安全用于体内使用的纳米颗粒也有可能增强天然化合物的生物利用度或用于药物输送。