IMMUNE REGULATION AND VACCINE DEVELOPMENT IN LEISHMANIASIS

利什曼病的免疫调节和疫苗开发

• 批准号: 8745304
• 负责人: David Sacks
• 金额: $ 72.5万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 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/8745304
• 关键词: Address; Adoptive Transfer; Agonist; Animal Model; Animals; Antigen Presentation; Antigens; Biological Assay; Bite; Blood Cells; C57BL/6 Mouse; CD4 Positive T Lymphocytes; CD44 gene; Caspase-1; Cell Count; Cells; Chronic; Chronic Phase; Clinical; Clinical Trials; Control Animal; Cutaneous; Defect; Dermal; Development; Disease; Drug or chemical Tissue Distribution; Ear; Effector Cell; Emulsions; Environment; Epitopes; Failure; Frequencies; Growth; Healed; Home environment; Host Defense; Human; Immune; Immune response; Immunity; Immunosuppressive Agents; India; Individual; Infection; Interleukin-10; Interleukin-17; Leishmania; Leishmaniasis; Lesion; Life; Lipid A; Major Histocompatibility Complex; Mediating; Mediator of activation protein; Membrane Glycoproteins; Memory; Messenger RNA; Modeling; Monitor; Mus; Natural Immunity; Nature; Needles; Oils; Parasites; Pathology; Patients; Peptides; Peripheral Blood Mononuclear Cell; Phase III Clinical Trials; Phenotype; Plasma; Polyproteins; Process; Production; Proliferating; Recruitment Activity; Regulation; Regulatory T-Lymphocyte; Research Design; Resistance; Role; SELL gene; Sand Flies; Series; Site; Skin; Specificity; Speed; Spleen; T cell response; T-Cell Receptor; T-Lymphocyte; TLR4 gene; Testing; Th1 Cells; Tissues; Vaccinated; Vaccines; Visceral; Visceral Leishmaniasis; Water; Whole Blood; acquired immunity; base; cytokine; exhaust; healing; immune clearance; killings; microbial; migration; mouse model; neutrophil; pathogen; prevent; response; transmission process; vaccination strategy; vaccine candidate; vaccine development; vaccine efficacy; vaccine evaluation

Many experimental vaccines have been developed that show promise in animal models of cutaneous or visceral forms of leishmaniasis, but an effective human vaccine still does not exist. In a series of phase III clinical trials in which killed Leishmania vaccines were tested in individuals exposed to infected sand fly bites in the field, no protection was observed. Remarkably, none of the candidate vaccines tested in animal models have been evaluated under experimental conditions using infected sand flies, and we addressed the possibility that vaccine failure in clinical trials may reflect the more stringent conditions of natural sand fly challenge. We compared needle and infected sand fly challenge to evaluate the efficacy of the only defined candidate vaccine currently in clinical trial, comprised of polyproteins (either KSAC or L110f), containing multiple antigenic epitopes from Leishmania delivered in a stable emulsion (SE) (water in oil) with glucopyranosyl lipid A (GLA), a TLR4 agonist suitable for use in people. Polyprotein-vaccinated mice had a 60-fold increase in CD4+ IFNg+ T cell numbers versus control animals at 2 wk postneedle inoculation of L. major, and this correlated with a 100-fold reduction in parasite load. By contrast, following challenge by infected sand fly bite, polyprotein-vaccinated animals had comparable parasite loads, greater numbers of neutrophils at the challenge site, and reduced CD4+IFNg+/IL-17+ ratios versus non-vaccinated controls. Importantly, mice with a healed primary infection were solidly protected against infected sand fly challenge that was associated with the speed with which effector cells appeared at a site of challenge, providing an immediate burst of effector cytokines that may be required to counteract the down-modulatory environment created by the highly localized, neutrophil-dominated, response to sand fly bite. We employed Ly6C, a T-bet regulated, GPI-anchored, surface glycoprotein expressed on T-bethi CD4+ T cells, to phenotype the rapidly recruited cells, and found that pre-existing, short-lived, CD44+CD62L- T-bet+Ly6C+ effector (TEFF) cells, not memory or memory-derived cells, mediate concomitant immunity in healed mice. Upon adoptive transfer and challenge, non-dividing Ly6C+ TEFF cells preferentially homed to the skin, released IFNg and conferred protection. Despite being short-lived, Ly6C+ TEFF cells were maintained at high frequencies in the mice with healed primary infection, presumably as a consequence of the persistent infection in these mice. The lack of effective vaccines against Leishmaniasis may be because protection against these infections requires concomitant immunity mediated by pre-existing TEFF cells, not memory cells, and is therefore not amenable to conventional, memory inducing, vaccination strategies. As indicated above, primary L. major infection typically produces cutaneous lesions that heal but that harbor persistent parasites. While the opposing roles of CD4+ T cell-derived IFNg and IL-10 in promoting parasite killing and persistence have been well established, how these responses develop from nave precursors has not been directly monitored throughout the course of infection. We used peptide:Major Histocompatibility Complex II (pMHCII) tetramers to investigate the endogenous, parasite-specific primary CD4+ T cell response to L. major in mice resistant to infection, and applied this approach to enumerate the expansion, contraction, tissue distribution, and function of parasite-specific CD4+ T cells throughout the course of the infection. Maximal frequencies of IFNg+ CD4+ T cells were observed in the spleen and infected ears within a month after infection and were maintained into the chronic phase. In contrast, peak frequencies of IL-10+CD4+ T cells emerged within 2 weeks of infection, persisted into the chronic phase, and accumulated in the infected ears but not the spleen, via a process that depended on local antigen presentation. T helper type-1 (Th1) cells, not Foxp3+ regulatory T cells, were the chief producers of IL-10 and were not exhausted. Therefore, tracking antigen-specific CD4+ T cells revealed that IL-10 production by Th1 cells is not due to persistent T cell antigen receptor stimulation, but rather driven early and sustained locally by antigen encounter at the site of infection. In addition to studying the mechanisms underlying parasite persistence in healed mice, mouse models are also being used to study non-healing forms of disease. Infection of C57BL/6 mice with most L. major strains results in a healing lesion with minimal pathology at the site of inoculation in the skin. By contrast, using a strain of L. major (Lm Sd) isolated from a patient with chronic cutaneous lesions, C57BL/6 mice also fail to heal their dermal lesions or effectively control tissue parasite burden despite a strong and polarized Th1 response. In studies designed to identify the earliest cells and mediators that precede and promote the severe pathology, we detected elevated levels of IL-1b mRNA and IL-1b+ cells in the inoculation site 3-4 weeks post-infection, followed by a neutrophil infiltrate that persisted until the onset of the pathology. Whereas no phenotype was observed in IL-17 deficient mice, IL-1R deficient mice, as well as IL-1b, ASC, and caspase-1/11 deficient mice, each showed minimal pathology and healed their Lm Sd infection. These studies are unique in the innate immunity field in identifying inflammasome dependent IL-1b as preventing rather than promoting host defense against a microbial pathogen. The mechanisms underlying the failure to control the growth and systemic spread of Leishmania parasites in human visceral leishmaniasis (VL) are not well understood. A key immunological feature of VL is the inability of peripheral blood mononuclear cells (PBMCs) to proliferate or to produce IFNg in response to leishmanial antigens. IL-10 has been implicated in the suppression of antigen-specific T cell responses in human VL based on the elevated levels of IL-10 observed in plasma and lesional tissue, and its role in preventing clearance of L. donovani in murine models of VL. In unexpected findings from our recent studies we were able to show that in sharp contrast to assays employing PBMCs, a cytokine release assay involving L. donovani antigen-stimulated whole-blood cells was able to detect the secretion of IFNg by cells from the majority of patients with active VL. In 35 patients with active VL, 80% secreted high levels of IFNg as compared to 85% of cured VL patients, and 24% of EHCs with presumed subclinical infections. The findings do not support a severe Th1 response defect in kala-azar. Importantly, only the whole blood cells from patients with active VL also secreted IL-10, which better reflects the response that distinguishes individuals with active disease from cured or subclinically infected, immune individuals. More generally, the findings reveal that PBMCs may not accurately reflect the immune competency of peripheral blood cells.

已经开发出许多实验性疫苗，在皮肤或内脏形式的利什曼病动物模型中显示出希望，但仍然不存在有效的人类疫苗。在一系列 III 期临床试验中，在现场暴露于受感染白蛉叮咬的个体中测试了灭活利什曼原虫疫苗，但没有观察到任何保护作用。 值得注意的是，在动物模型中测试的候选疫苗都没有在使用受感染的白蛉的实验条件下进行评估，我们解决了临床试验中疫苗失败可能反映出自然白蛉挑战的更严格条件的可能性。 我们比较了针头和受感染的沙蝇攻击，以评估目前临床试验中唯一确定的候选疫苗的功效，该疫苗由多蛋白（KSAC 或 L110f）组成，含有来自利什曼原虫的多个抗原表位，以稳定乳液（SE）（水在油）与吡喃葡萄糖基脂质 A (GLA)，一种适合人类使用的 TLR4 激动剂。 在接种 L. Major 后 2 周，多蛋白疫苗接种小鼠的 CD4+ IFNg+ T 细胞数量比对照动物增加 60 倍，这与寄生虫负载减少 100 倍相关。 相比之下，在受到受感染白蛉叮咬攻击后，与未接种疫苗的对照相比，多蛋白疫苗接种的动物具有相当的寄生虫负载、攻击部位的中性粒细胞数量较多，并且 CD4+IFNg+/IL-17+ 比率降低。 重要的是，原发性感染治愈的小鼠得到了牢固的保护，免受感染白蛉的攻击，这与效应细胞出现在攻击部位的速度有关，从而提供了抵消下调调节可能需要的效应细胞因子的立即爆发。由高度局部化、中性粒细胞主导、对白蛉叮咬的反应所创造的环境。 我们使用 Ly6C（一种 T-bet 调节的、GPI 锚定的、在 T-bethi CD4+ T 细胞上表达的表面糖蛋白）来对快速募集的细胞进行表型分析，并发现预先存在的、短寿命的 CD44+CD62L-T-bet +Ly6C+效应（TEFF）细胞，而不是记忆或记忆衍生细胞，在治愈小鼠中介导伴随免疫。在过继转移和攻击后，非分裂的 Ly6C+ TEFF 细胞优先归巢于皮肤，释放 IFNg 并提供保护。尽管寿命短暂，但在原发感染治愈的小鼠中，Ly6C+ TEFF 细胞仍保持高频率，这可能是这些小鼠持续感染的结果。缺乏针对利什曼病的有效疫苗可能是因为针对这些感染的保护需要由预先存在的 TEFF 细胞而不是记忆细胞介导的伴随免疫，因此不适合传统的记忆诱导疫苗接种策略。 如上所述，原发性大型利斯特氏菌感染通常会产生可愈合但含有持续寄生虫的皮肤损伤。 虽然 CD4+ T 细胞衍生的 IFNg 和 IL-10 在促进寄生虫杀伤和持久性方面的相反作用已得到充分证实，但这些反应如何从幼稚前体发展而来，尚未在整个感染过程中直接监测。 我们使用肽：主要组织相容性复合体 II (pMHCII) 四聚体来研究抗感染小鼠中对 L. Major 的内源性、寄生虫特异性初级 CD4+ T 细胞反应，并应用这种方法来列举扩张、收缩、组织分布和寄生虫特异性 CD4+ T 细胞在整个感染过程中的功能。 感染后一个月内，在脾脏和感染耳朵中观察到 IFNg+ CD4+ T 细胞的最大频率，并维持到慢性期。 相比之下，IL-10+CD4+ T 细胞的峰值频率在感染后 2 周内出现，持续进入慢性期，并通过依赖于局部抗原呈递的过程在受感染的耳朵而不是脾脏中积累。 1 型辅助 T (Th1) 细胞（而非 Foxp3+ 调节性 T 细胞）是 IL-10 的主要产生者，并且并未耗尽。 因此，追踪抗原特异性 CD4+ T 细胞表明，Th1 细胞产生 IL-10 并不是由于持续的 T 细胞抗原受体刺激，而是由感染部位的抗原相遇引起的早期驱动和局部持续。 除了研究治愈小鼠体内寄生虫持续存在的机制外，小鼠模型还被用于研究非治愈疾病。 用大多数主要菌株感染 C57BL/6 小鼠会导致皮肤接种部位出现愈合损伤，且病变程度最小。相比之下，使用从患有慢性皮肤损伤的患者中分离出的 L. Major (Lm Sd) 菌株，尽管有强烈且极化的 Th1 反应，但 C57BL/6 小鼠也无法治愈其皮肤损伤或有效控制组织寄生虫负荷。 在旨在识别先于并促进严重病理的最早细胞和介质的研究中，我们在感染后 3-4 周检测到接种部位的 IL-1b mRNA 和 IL-1b+ 细胞水平升高，随后出现中性粒细胞浸润，一直持续到病理发生。 尽管在 IL-17 缺陷型小鼠、IL-1R 缺陷型小鼠以及 IL-1b、ASC 和 caspase-1/11 缺陷型小鼠中未观察到表型，但每种小鼠均表现出最小的病理并治愈了 Lm Sd 感染。这些研究在先天免疫领域是独一无二的，它们确定了炎症小体依赖性 IL-1b 可以预防而不是促进宿主对微生物病原体的防御。 人类内脏利什曼病（VL）中利什曼原虫生长和系统性传播未能得到控制的机制尚不清楚。 VL 的一个关键免疫学特征是外周血单核细胞 (PBMC) 无法增殖或产生 IFNg 来响应利什曼原虫抗原。根据血浆和病变组织中观察到的 IL-10 水平升高，以及 IL-10 在 VL 小鼠模型中阻止杜氏乳杆菌清除的作用，IL-10 与人 VL 中抗原特异性 T 细胞反应的抑制有关。 我们最近研究的意外发现表明，与使用 PBMC 的测定形成鲜明对比，涉及杜氏乳杆菌抗原刺激的全血细胞的细胞因子释放测定能够检测来自大多数细胞的 IFNg 分泌。活动性 VL 患者。 在 35 名活动性 VL 患者中，80% 的人分泌高水平的 IFNg，而治愈的 VL 患者的分泌水平为 85%，而 EHC 的推测为亚临床感染的比例为 24%。研究结果并不支持黑热病存在严重的 Th1 反应缺陷。 重要的是，只有活动性 VL 患者的全血细胞也会分泌 IL-10，这更好地反映了将活动性疾病个体与已治愈或亚临床感染的免疫个体区分开来的反应。 更一般地说，研究结果表明 PBMC 可能无法准确反映外周血细胞的免疫能力。