Development of a Vaccine for HIV-AIDS: Cellular Immunity

艾滋病毒/艾滋病疫苗的开发：细胞免疫

• 批准号: 10262216
• 负责人: Marjorie Robert-Guroff
• 金额: $ 34.34万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 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/10262216
• 关键词: Adaptive Immune System; Adenovirus Vector; Adenoviruses; Alveolar Macrophages; Animals; Antibodies; Antibody Response; Antigens; Attenuated Live Virus Vaccine; Autologous; Autopsy; B-Lymphocytes; Blood; Bronchoalveolar Lavage; CD6 antigen; CD8-Positive T-Lymphocytes; Cell Line; Cells; Cellular Immunity; Chronic; Coculture Techniques; Communicable Diseases; Data; Dendritic Cells; Development; Disease Progression; Disease susceptibility; Dose; Drops; Epithelial Cells; Exhibits; Exposure to; Frequencies; Functional disorder; Genes; Goals; HIV; HIV Infections; HIV vaccine; Human; Immune; Immune response; Immunity; Immunization; Immunize; Immunologic Memory; Impairment; In Vitro; Individual; Infection; Interferon Type II; Interleukin-17; Interleukin-6; Intramuscular; Knowledge; Langerhans cell; Lead; Life; Lung; Lung diseases; Lymphoid Cell; Lymphoid Tissue; Macaca; Macaca mulatta; Macrophage Activation; Measles; Mediating; Memory; Modeling; Mucous Membrane; Myelogenous; Natural Immunity; Natural Killer Cells; PD-1 blockade; Pathogenicity; Pathway interactions; Phagocytes; Phagocytosis; Phenotype; Phosphoric Monoester Hydrolases; Plasma; Play; Poliomyelitis; Population; Production; Property; RANTES; Recombinants; Regimen; Regulation; Risk; Role; SIV; SIV Vaccines; Site; Smallpox Vaccine; Splenocyte; Surface; T memory cell; T-Lymphocyte; TNF gene; Therapeutic; Time; Tissues; Upper respiratory tract; Vaccination; Vaccines; Vagina; Viral Load result; Viremia; Virus; Yellow Fever; antibody-dependent cell cytotoxicity; base; cell type; cervicovaginal; chemokine; chronic infection; cytokine; cytotoxicity; design; effector T cell; env Gene Products; immune activation; improved; in vivo; insight; lymph nodes; macrophage; mucosal site; mucosal vaccination; mutant; natural killer cell protein 44-kDa; overexpression; pre-clinical; programmed cell death protein 1; protective efficacy; receptor; recombinant adenovirus; recruit; rectal; response; simian human immunodeficiency virus; targeted treatment; therapy development; transmission process; trend; vaccination strategy; vaccine development; vaccine trial; vaccine-induced immunity; vector

We are pursuing an HIV vaccine approach based on replication-competent Adenovirus (Ad)-recombinants. The rationale for this strategy is based on the fact that live attenuated vaccines historically have been the most protective, eliciting essentially life-long immunity. Examples include vaccines for smallpox, polio, measles, and yellow fever. We conduct pre-clinical vaccine studies in rhesus macaques and challenge with SIV or SHIV (a chimeric SIV virus containing an HIV envelope), viruses that model HIV-infection of humans. We use a prime-boost strategy, first immunizing mucosally with a replicating adenovirus (Ad) vector carrying an HIV/SIV gene(s) followed by a boosting intramuscularly with HIV/SIV envelope protein. Ad replicates in epithelial cells that line mucosal inductive sites, and therefore elicits strong, persistent cellular immunity at mucosal effector sites as well as in the blood. In studying vaccine-induced cellular immunity, we focused on several specific cell types. Natural killer (NK) cells have a rapid response potential, can kill target cells directly, and mediate antibody-dependent effector functions such as antibody-dependent cellular cytotoxicity (ADCC) which has been shown to contribute to protective efficacy. They can also persist for long periods of time in vivo and have the capacity to establish immunologic memory. We investigated NK cell and innate lymphoid cell (ILC) dynamics and function in rhesus macaque rectal tissue and blood following our vaccination strategy and subsequent repeated low-dose intravaginal SIV exposures. Mucosal memory-like NK and ILC subsets in rectal and vaginal tissues of chronically infected macaques were also evaluated. Mucosal IL-17+NKp44+ ILCs were previously associated with a decreased risk of SIV acquisition and IFN-g+ double-negative (DN) ILCs with an increased risk of acquisition. We confirmed and extended these observations, showing that not only did NKp44+ and DN cells exhibit similar associations, but the respective effects continued past SIV infection, contributing to regulation of viral loads. The data suggested that mucosal NKp44+ and DN ILC subsets play protective and pathogenic roles contributing to viremia control and disease progression, respectively. We also identified gamma-chain deficient, Syk-deficient memory-like delta-gamma NK cells in rectal, endocervical, ectocervical, and vaginal tissues obtained at necropsy from the rhesus macaques. The overall frequency of the mucosal delta-gamma cell populations was higher in the mucosal tissues of macaques with low VLs compared to macaques with high VLs suggesting they might contribute to viremia control. Overall, our results suggested that vaccines that favor expansion of NKp44+ delta-gamma cells, while avoiding DN delta-gamma cells, might be beneficial for control of SIV/HIV infection. Dendritic cells (DCs) orchestrate both innate and adaptive effector immune cell responses. We investigated their role in vaccine-induced immunity in rhesus macaques. Following mucosal immunizations with replicating Ad type 5 host range mutant (Ad5hr)-SIV recombinants, DC subsets and their activation were examined in rectal tissue, blood, and lymph nodes (LN) at 3 timepoints after each immunization. Plasmacytoid DCs, myeloid DCs, and Langerhans cells were significantly increased in the rectal mucosa, but only myeloid DCs were significantly increased in blood post-immunizations. All rectal DC subsets showed increased frequencies of cells expressing activation markers and cytokines post-immunization, blood DCs showed mixed results, and LN DCs showed few changes. Rectal DCs responded strongly to the vector rather than expressed SIV antigens, but rectal DC frequencies positively correlated with induced rectal antigen-specific memory T and B cells. In vitro co-cultures confirmed that rectal Ad-SIV DCs induced proliferation and antigen-specific cytokine production by autologous naive T cells. These results highlighted the rapid response of DCs to Ad immunization and their role in mucosal immune activation and identified initial cellular mechanisms of the replicating Ad-SIV vaccine in the rhesus macaque model. Alveolar macrophages (AMs) play a critical role in lung innate immunity. Using the SIV rhesus macaque model, we investigated the effect of SIV infection on the phenotypic and functional properties of AMs. AM expression of proinflammatory cytokines TNF-a, IL-6, IL-1B, and chemokine RANTES drastically increased 2 weeks post-infection (wpi) compared to AMs of uninfected macaques but dropped significantly with progression to chronic infection. Phagocytic activity of AMs 2-and 4-wpi was elevated compared to AMs of uninfected animals but again significantly decreased by 12-wpi. By 20-wpi the ability of AMs from chronically infected animals to perform SIV-specific antibody-dependent phagocytosis (ADP) was also diminished. PD-1 was expressed on AMs and showed a strong trend toward correlation with plasma viral load, indicating that similar to over-expression on T-cells, PD-1 expression on AMs may be associated with disease progression. Importantly, blockade of PD-1 improved phagocytic function. These findings provide new insight into the dynamics of SIV infection leading to AM dysfunction and alteration of pulmonary innate immunity and suggest new pathways to exploit in developing therapies targeting pulmonary disease susceptibility in HIV-infected individuals. We furthered these studies by evaluating cervicovaginal macrophages (CVM) in rhesus macaques over the course of an SIV vaccine regimen. As one of the first cells exposed to SIV, their interaction with the virus might lead to activation and recruitment of additional susceptible target cells but might also provide important early protection. Knowledge of their response to mucosal vaccination is critical for design and development of improved vaccine strategies. As our mucosal priming immunizations were administered to the upper respiratory tract, we also investigated alveolar macrophages (AM) over the course of immunization. We found that while vaccination induced chemokine responses by CVM, recruitment of susceptible cells did not result. Further, induction of FcgammaRIII expression on CVM was associated with delayed SIV acquisition. At the same time vaccination induced AM activation and phagocytic activity along with expression of factors associated with B cell help. Overall, these findings suggest that vaccine enhancement of macrophage responses may lead to greater efficacy. In view of the association of PD-1-expressing cells with increased viral loads we investigated other cell populations expressing this molecule. We identified a dysfunctional CD8+ T cell population co-expressing CD6 and PD-1 in lymphoid tissues and bronchoalveolar lavage of rhesus macaques chronically infected with SIV. This cell population was expanded compared to uninfected cells and displayed impaired proliferation, cytokine secretion, and cytotoxicity. The frequency of the cells positively correlated with viremia. The cells expressed elevated levels of the inhibitory receptor LAG-3 and SHP-2 phosphatase compared to CD6-PD-1+ CD8+ T cells suggesting a mechanism by which CD6 might induce T cell dysfunction. Combined targeting of CD6 and PD-1 in vitro revived CD8+ T cell effector function of splenocytes of infected macaques suggesting a potential therapeutic strategy.

我们正在寻求一种基于具有复制能力的腺病毒 (Ad) 重组体的 HIV 疫苗方法。这一策略的基本原理是基于这样一个事实：减毒活疫苗历来是最具保护性的，基本上能产生终身免疫。例如天花、脊髓灰质炎、麻疹和黄热病疫苗。我们在恒河猴中进行临床前疫苗研究，并用 SIV 或 SHIV（一种含有 HIV 包膜的嵌合 SIV 病毒）进行攻击，这些病毒模拟人类 HIV 感染。我们采用初免-加强策略，首先用携带 HIV/SIV 基因的复制腺病毒 (Ad) 载体进行粘膜免疫，然后用 HIV/SIV 包膜蛋白进行肌内加强免疫。 Ad 在粘膜诱导位点的上皮细胞中复制，因此在粘膜效应位点以及血液中引发强烈、持久的细胞免疫。在研究疫苗诱导的细胞免疫时，我们重点关注几种特定的细胞类型。自然杀伤（NK）细胞具有快速反应潜力，可以直接杀死靶细胞，并介导抗体依赖性效应功能，例如抗体依赖性细胞毒性（ADCC），这已被证明有助于保护功效。它们还可以在体内长期存在，并具有建立免疫记忆的能力。根据我们的疫苗接种策略和随后重复的低剂量阴道内 SIV 暴露，我们研究了恒河猴直肠组织和血液中 NK 细胞和先天淋巴细胞 (ILC) 的动态和功能。还评估了慢性感染猕猴直肠和阴道组织中的粘膜记忆样 NK 和 ILC 亚群。粘膜 IL-17+NKp44+ ILC 先前被认为与 SIV 获得风险降低相关，而 IFN-g+ 双阴性 (DN) ILC 与获得风险增加相关。我们证实并扩展了这些观察结果，表明 NKp44+ 和 DN 细胞不仅表现出相似的关联，而且各自的影响在 SIV 感染后持续存在，有助于病毒载量的调节。数据表明，粘膜 NKp44+ 和 DN ILC 亚群分别发挥保护性和致病性作用，有助于病毒血症控制和疾病进展。我们还在恒河猴尸检时获得的直肠、宫颈内膜、宫颈外膜和阴道组织中发现了伽马链缺陷、Syk 缺陷记忆样 δ-伽玛 NK 细胞。与高 VL 的猕猴相比，低 VL 的猕猴粘膜组织中粘膜 δ-γ 细胞群的总体频率较高，表明它们可能有助于病毒血症的控制。总体而言，我们的结果表明，有利于 NKp44+ δ-γ 细胞扩增，同时避免 DN δ-γ 细胞的疫苗可能有利于控制 SIV/HIV 感染。树突状细胞 (DC) 协调先天性和适应性效应免疫细胞反应。我们研究了它们在恒河猴疫苗诱导免疫中的作用。用复制型Ad 5型宿主范围突变体(Ad5hr)-SIV重组体进行粘膜免疫后，在每次免疫后的3个时间点检查直肠组织、血液和淋巴结(LN)中的DC亚群及其活化。直肠粘膜中浆细胞样DC、髓样DC和朗格汉斯细胞显着增加，但免疫后血液中仅髓样DC显着增加。所有直肠 DC 亚群均显示免疫后表达激活标记和细胞因子的细胞频率增加，血液 DC 显示混合结果，而 LN DC 显示很少变化。直肠 DC 对载体反应强烈，而不是表达 SIV 抗原，但直肠 DC 频率与诱导的直肠抗原特异性记忆 T 和 B 细胞呈正相关。体外共培养证实，直肠 Ad-SIV DC 可诱导自体幼稚 T 细胞增殖和产生抗原特异性细胞因子。这些结果强调了 DC 对 Ad 免疫的快速反应及其在粘膜免疫激活中的作用，并确定了在恒河猴模型中复制 Ad-SIV 疫苗的初始细胞机制。肺泡巨噬细胞（AM）在肺部先天免疫中发挥着关键作用。使用 SIV 恒河猴模型，我们研究了 SIV 感染对 AMs 表型和功能特性的影响。与未感染猕猴的 AM 相比，促炎细胞因子 TNF-a、IL-6、IL-1B 和趋化因子 RANTES 的 AM 表达在感染后 2 周 (wpi) 急剧增加，但随着进展为慢性感染而显着下降。与未感染动物的 AM 相比，感染后第 2 周和第 4 周 AM 的吞噬活性升高，但在感染后第 12 周再次显着降低。到感染后 20 周时，来自慢性感染动物的 AM 执行 SIV 特异性抗体依赖性吞噬作用 (ADP) 的能力也减弱了。 PD-1 在 AM 上表达，并显示出与血浆病毒载量相关的强烈趋势，表明与 T 细胞上的过度表达类似，AM 上的 PD-1 表达可能与疾病进展相关。重要的是，阻断 PD-1 可以改善吞噬功能。这些发现为 SIV 感染导致 AM 功能障碍和肺部先天免疫改变的动态提供了新的见解，并提出了开发针对 HIV 感染者肺部疾病易感性的治疗方法的新途径。我们通过评估 SIV 疫苗方案过程中恒河猴的子宫颈阴道巨噬细胞 (CVM) 来进一步开展这些研究。作为第一批暴露于 SIV 的细胞之一，它们与病毒的相互作用可能会导致其他易感靶细胞的激活和招募，但也可能提供重要的早期保护。了解它们对粘膜疫苗接种的反应对于设计和开发改进的疫苗策略至关重要。由于我们的粘膜引发免疫是针对上呼吸道进行的，因此我们还在免疫过程中研究了肺泡巨噬细胞（AM）。我们发现，虽然疫苗接种通过 CVM 诱导趋化因子反应，但并没有导致易感细胞的募集。此外，CVM 上 FcgammaRIII 表达的诱导与 SIV 获得延迟相关。同时，疫苗接种诱导 AM 激活和吞噬细胞活性，以及​​与 B 细胞相关的因子表达。总的来说，这些发现表明疫苗增强巨噬细胞反应可能会带来更大的功效。鉴于表达 PD-1 的细胞与病毒载量增加之间的关联，我们研究了表达该分子的其他细胞群。我们在慢性感染 SIV 的恒河猴的淋巴组织和支气管肺泡灌洗液中发现了共表达 CD6 和 PD-1 的功能失调的 CD8+ T 细胞群。与未感染的细胞相比，该细胞群有所扩大，并表现出增殖、细胞因子分泌和细胞毒性受损。细胞的频率与病毒血症呈正相关。与 CD6-PD-1+ CD8+ T 细胞相比，这些细胞表达的抑制性受体 LAG-3 和 SHP-2 磷酸酶水平升高，这表明 CD6 可能诱导 T 细胞功能障碍的机制。体外联合靶向 CD6 和 PD-1 可恢复受感染猕猴脾细胞的 CD8+ T 细胞效应功能，这表明了一种潜在的治疗策略。

项目成果

HIV-1 CD4-induced (CD4i) gp120 epitope vaccines promote B and T-cell responses that contribute to reduced viral loads in rhesus macaques.

HIV-1 CD4诱导的（CD4I）GP120表位疫苗促进B和T细胞反应，从而导致恒河猕猴病毒载量减少。

• DOI: 10.1016/j.virol.2014.10.001
• 发表时间: 2014-12
• 期刊: VIROLOGY
• 影响因子: 3.7
• 作者: Thomas, Michael A.;Tuero, Iskra;Demberg, Thorsten;Vargas-Inchaustegui, Diego A.;Musich, Thomas;Xiao, Peng;Venzon, David;LaBranche, Celia;Montefiori, David C.;DiPasquale, Janet;Reed, Steven G.;DeVico, Anthony;Fouts, Timothy;Lewis, George K.;Gallo, Robert C.;Robert-Guroff, Marjorie
• 通讯作者: Robert-Guroff, Marjorie

Phenotypic and Functional Characterization of Circulatory, Splenic, and Hepatic NK Cells in Simian Immunodeficiency Virus-Controlling Macaques.

• DOI: 10.4049/jimmunol.1700586
• 发表时间: 2017-11-01
• 期刊: Journal of immunology (Baltimore, Md. : 1950)
• 作者: Vargas-Inchaustegui DA;Helmold Hait S;Chung HK;Narola J;Hoang T;Robert-Guroff M
• 通讯作者: Robert-Guroff M

Replicating Adenovirus-SIV Immunization of Rhesus Macaques Induces Mucosal Dendritic Cell Activation and Function Leading to Rectal Immune Responses.

恒河猴的复制腺病毒-SIV 免疫诱导粘膜树突状细胞激活和功能，导致直肠免疫反应。

• DOI: 10.3389/fimmu.2019.00779
• 发表时间: 2019
• 期刊: Frontiers in immunology
• 影响因子: 7.3
• 作者: Ko,Eun-Ju;HelmoldHait,Sabrina;Enyindah-Asonye,Gospel;Rahman,MohammadArif;Hoang,Tanya;Robert-Guroff,Marjorie
• 通讯作者: Robert-Guroff,Marjorie

Differential Effect of Mucosal NKp44+ Innate Lymphoid Cells and Δγ Cells on Simian Immunodeficiency Virus Infection Outcome in Rhesus Macaques.

粘膜 NKp44 先天淋巴细胞和 γ 细胞对恒河猴免疫缺陷病毒感染结果的差异作用。

• DOI: 10.4049/jimmunol.1900572
• 发表时间: 2019
• 期刊: Journal of immunology (Baltimore, Md. : 1950)
• 作者: Rahman,MohammadArif;Ko,Eun-Ju;Enyindah-Asonye,Gospel;HelmoldHait,Sabrina;Hogge,Christopher;Hunegnaw,Ruth;Venzon,DavidJ;Hoang,Tanya;Robert-Guroff,Marjorie
• 通讯作者: Robert-Guroff,Marjorie