Development of a Vaccine for HIV-AIDS: Cellular Immunity

我们正在寻求一种基于复制能力腺病毒（Ad）重组的HIV疫苗方法。这一战略的基本原理是基于这样一个事实，即减毒活疫苗在历史上是最具保护性的，基本上可以产生终身免疫。例子包括天花、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，并使用SIV或SHIV（一种含有HIV包膜的嵌合SIV病毒）进行挑战，这些病毒模拟人类HIV感染。我们采用一种初始增强策略，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包膜蛋白进行肌肉增强。Ad在粘膜诱导部位的上皮细胞中复制，因此在粘膜效应部位和血液中引发强烈、持久的细胞免疫。在研究疫苗诱导的细胞免疫时，我们着重于几种特定的细胞类型。自然杀伤细胞（NK）具有快速的反应潜力，可以直接杀死靶细胞，并介导抗体依赖性效应功能，如抗体依赖性细胞毒性（ADCC），已被证明有助于保护功效。它们也可以在体内持续很长一段时间，并具有建立免疫记忆的能力。我们研究了NK细胞和先天淋巴样细胞（ILC）在恒河猴直肠组织和血液中的动态和功能，在我们的疫苗接种策略和随后的重复低剂量阴道内SIV暴露。我们还对慢性感染猕猴直肠和阴道组织中的粘膜记忆样NK和ILC亚群进行了评估。粘膜IL-17+NKp44+ ILCs先前与SIV获得风险降低相关，IFN-g+双阴性（DN） ILCs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，有助于调节病毒载量。这些数据表明，粘膜NKp44+和DN ILC亚群分别在病毒血症控制和疾病进展中发挥保护和致病作用。我们还在恒河猴的直肠、宫颈内、宫颈外和阴道组织中发现了γ链缺陷、syk缺陷的记忆样δ - γ NK细胞。与VLs高的猕猴相比，VLs低的猕猴粘膜组织中δ - γ细胞群的总体频率更高，这表明它们可能有助于病毒血症的控制。总的来说，我们的结果表明，有利于NKp44+ δ - γ细胞扩增而避免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Ad5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个时间点检测直肠组织、血液和淋巴结（LN）中的DC亚群及其激活情况。浆细胞样树突状细胞、髓细胞样树突状细胞和朗格汉斯细胞在直肠粘膜中显著增加，但免疫后血液中只有髓细胞样树突状细胞显著增加。所有直肠DC亚群显示免疫后表达活化标记物和细胞因子的细胞频率增加，血液DC显示混合结果，LN DC几乎没有变化。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诱导自体幼稚T细胞增殖和抗原特异性细胞因子的产生。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，并确定了复制Ad- siv疫苗在恒河猴模型中的初始细胞机制。肺泡巨噬细胞（AMs）在肺部先天免疫中起着至关重要的作用。利用SIV恒河猴模型，研究SIV感染对AMs表型和功能特性的影响。与未感染AM的猕猴相比，AM的促炎细胞因子TNF-a、IL-6、IL-1B和趋化因子RANTES的表达在感染后2周（wpi）急剧增加，但随着慢性感染的进展而显著下降。与未感染动物的AMs相比，AMs 2-和4-wpi的吞噬活性升高，但再次显著降低12-wpi。到20wpi时，来自慢性感染动物的AMs进行siv特异性抗体依赖性吞噬（ADP）的能力也减弱。PD-1在AMs上表达，并表现出与血浆病毒载量密切相关的趋势，表明与t细胞上的过表达类似，PD-1在AMs上的表达可能与疾病进展有关。重要的是，阻断PD-1可改善吞噬功能。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力学提供了新的见解，并为开发针对hiv感染者肺部疾病易感性的治疗方法提供了新的途径。我们通过在SIV疫苗治疗过程中评估恒河猴宫颈阴道巨噬细胞（CVM）来进一步研究这些研究。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导致激活和募集额外的易感靶细胞，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。当我们的粘膜启动免疫被施用于上呼吸道时，我们也研究了免疫过程中的肺泡巨噬细胞（AM）。我们发现，虽然疫苗接种诱导了CVM的趋化因子反应，但没有导致易感细胞的募集。此外，CVM上FcgammaRIII表达的诱导与SIV获得延迟有关。同时，疫苗接种诱导AM活化和吞噬活性，并伴有与B细胞帮助相关因子的表达。总之，这些发现表明，疫苗增强巨噬细胞反应可能导致更大的疗效。鉴于pd -1表达细胞与病毒载量增加的关联，我们研究了其他表达该分子的细胞群。我们在慢性SIV感染恒河猴的淋巴组织和支气管肺泡灌洗液中发现了功能失调的CD8+ T细胞群共表达CD6和PD-1。与未感染的细胞相比，该细胞群扩大，并表现出增殖、细胞因子分泌和细胞毒性受损。细胞出现频率与病毒血症呈正相关。与CD6- pd -1+ CD8+ T细胞相比，这些细胞表达了更高水平的抑制受体LAG-3和SHP-2磷酸酶，提示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恢复感染猕猴脾细胞的CD8+ T细胞效应功能，提示一种潜在的治疗策略。

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.