IMMUNE REGULATION AND VACCINE DEVELOPMENT IN LEISHMANIASIS

利什曼病的免疫调节和疫苗开发

• 批准号: 7732462
• 负责人: David Sacks
• 金额: $ 65.59万
• 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/7732462
• 关键词: Accounting; Agar; Animals; Annexins; Antibodies; Antigens; Antimony; Apoptotic; Appearance; Aspirate substance; Attenuated Vaccines; Biological Response Modifiers; Biopsy; Bite; Blood; Bone Marrow; C57BL/6 Mouse; CD4 Positive T Lymphocytes; CD8B1 gene; Cells; Chronic; Clinical; Clinical Research; Coculture Techniques; Commit; Cutaneous; Cutaneous Leishmaniasis; Data; Data Set; Dendritic Cells; Deposition; Dermis; Development; Diagnosis; Disease; Dose; Employee Strikes; Equilibrium; Experimental Models; Failure; Follow-Up Studies; Functional disorder; Glycoproteins; Goals; Green Fluorescent Proteins; Growth; Healed; Homeostasis; Host resistance; Human; Immune; Immune response; Immunity; Immunologics; In Vitro; Inactivated Vaccines; India; Infection; Infection Control; Inflammation; Inflammatory; Inflammatory Response; Interferon Type II; Interleukin-10; Interleukin-17; Interleukin-4; Laboratories; Leishmania; Leishmaniasis; Lesion; Link; Mediating; Messenger RNA; Modeling; Muramidase; Mus; Nature; Needles; Neutrophil Infiltration; Numbers; Organ; Outcome; Parasites; Pathogenesis; Pathology; Patients; Peripheral Blood Mononuclear Cell; Plasma; Population; Predisposition; Production; Proliferating; Reaction; Recombinant Proteins; Regulation; Reporting; Research; Residual state; Residual volume; Resistance; Role; Saliva; Sampling; Sand Flies; Severity of illness; Site; Skin; Sorting - Cell Movement; Source; Spleen; Staging; Staining method; Stains; T-Lymphocyte; Testing; Th1 Cells; Time; Tissues; Transgenic Organisms; Tumor Necrosis Factor-alpha; Vaccines; Visceral; Visceral Leishmaniasis; Week; antimicrobial; base; cell type; cytokine; day; design; healing; human TNF protein; in vivo; interleukin-10 receptor; killings; lymph nodes; macrophage; mouse model; neutrophil; novel; pathogen; prevent; prophylactic; response; therapeutic vaccine; transmission process; uptake; vaccine development

Despite the encouraging results of non-living vaccines in experimental models, including defined DNA-based and recombinant protein-based vaccines, a safe, non-living vaccine has consistently failed to confer significant protection against cutaneous or visceral leishmaniasis in human trials. While there are apt to be many explanations for these discrepant outcomes, one key difference between the laboratory and field-based trials is the use of needle challenge to evaluate vaccines in animals, as compared to natural sand fly challenge in the human trials. Remarkably, no experimental vaccine has been evaluated by sand fly challenge in a controlled, laboratory setting. In addition to the delivery of infectious stage parasites into the dermis, infected sand flies also deposit pharmacologically active saliva, and parasite-derived secreted glycoproteins, each known to enhance the severity of disease when co-administered with parasites by needle. Furthermore, we have recently reported that sand fly transmission induces a qualitatively unique inflammatory response at the bite site that includes a dynamic recruitment of neutrophils and that these neutrophils markedly enhance the ability of parasites to establish primary infection. We have produced a clear set of data to indicate that the non-living vaccines that confer powerful protection against needle challenge are ineffective against sand fly challenge. The difference is not accounted for by the quality or dose of infectious stage parasites in the respective inocula. Neutrophil depletion following sand fly challenge rescued the ability of the killed vaccine to confer protection. Analysis at 2 weeks post-transmission revealed that neutrophil depletion resulted in increased Ag-specific interferon gamma (IFNg) production by CD4+ T cells. These findings demonstrate that the early recruitment and persistence of high numbers of neutrophils at the bite site seriously compromise the expression of secondary immunity in the skin. As neutrophils are the first cell type to take up Leishmania following transmission by bite, and as their specific depletion enhances host resistance to L. major, the possibility that infected neutrophils can encounter immature dendritic cells (iDCs) and compromise their function has been explored. Following i.d. inoculation of RFP-expressing L. major into lysozyme-GFP mice, the recovered GFP+ neutrophils were sorted into uninfected and infected populations, and co-cultured overnight with iDC. Following an additional sort of GFP+ and GFP- iDC, we investigated the priming capacity of these different populations of DC by co-culture with OT-I CD8+ T cells in the presence of soluble OVA. Uptake of infected neutrophils inhibited DC maturation, and completely eliminated their capacity to stimulate OT-I cells to proliferate and release IFNg. Their dysfunction was far more pronounced than the DC that had taken up uninfected neutrophils, a difference that correlated with the enhanced apoptotic status of the infected neutrophils, as determined by annexin staining. In vivo, neutrophil depletion increased the proliferation of adoptively transferred OT-I cells in response to infection by L. major transgenic parasites expressing OVA. These data demonstrate both a novel mechanism by which Leishmania parasites can induce immune dysregulation and a previously unrecognized role of infected neutrophils during the immune response to L. major. Endogenous IL-10 is a central mediator of immune homeostasis, necessary to keep in check the strong inflammatory reactions that can accompany the expression of anti-microbial immunity in local tissues. A consequence of the balance struck between host immunity and pathology can be chronic or persistent infection, and the absence of IL-10 has been shown to result in more efficient clearance of a variety of pathogens, including L. major. We recently reported on the inability of conventionally resistant C57BL/6 mice to successfully resolve infection by an isolate of L. major, despite a strong IFNg response by the host. Susceptibility was caused by antigen-specific IL-10 from CD4+ cells that were also producing IFNg. In followup studies, we have explored the role for IL-27 in the regulation of IL-10 from Th1 cells in Leishmaniasis. IL-27 was found to enhance IL-10 induction in L. major primed, committed Th1 cells in vitro. Cytokine analysis of CD4+ cells in the lesions and draining lymph nodes of infected IL-27R deficient (WSX-1-/-) mice revealed diminished IL-10 from IFNg+ CD4+ cells, which was accompanied by a reduction in total IFNg+ CD4+ cells and an increase in IL-4. Despite the inhibition of IL-10 from CD4+ cells, no significant change in parasite numbers was observed, due both to the dysregulated IL-4 and residual levels of IL-10. Strikingly, infected WSX-1-/- mice developed severe lesions that were associated with the appearance of IL-17+ CD4+ cells, demonstrating a function for IL-27 in blocking the development of inappropriate Th17 cells during L. major infection. The results demonstrate the pleiotropic effects that IL-27 has on L. major-driven Th1, Th2, and Th17 development, and reinforce its function as a key regulatory cytokine that controls the balance between immunity and pathology. The mechanisms underlying the failure to control the growth and systemic spread of Leishmania parasites in human visceral leishmaniasis (VL) are not well understood. Produced as a possible homeostatic mechanism to control persistent infection-induced inflammation, elevated levels of the suppressive cytokine IL-10 have repeatedly been observed in the spleen, bone marrow, PBMC, and plasma of VL patients with active disease. Within the parasitized spleen, the source ofthe elevated IL-10 mRNA was shown to be CD3+CD25-Foxp3- cells. Direct demonstration that IL-10 impairs antigen-specific IFNg responses and promotes parasite growth in a target organ such as the spleen would have strong relevance to the pathogenesis of VL. We have taken advantage of the presence of infected macrophages within the biopsied splenic cells as a readout for IL-10 function. The small volume of residual splenic aspirate obtained for diagnosis was split into two equal volumes, and cultured in the presence of anti-IL-10 antibodies or isotype control Ig. After 3 days, the number of viable parasites was quantified by serial dilution onto blood agar plates. Within the 18 paired samples for which parasites could be grown out, 14 had fewer parasites following the 3 day incubation with anti-IL-10, and in 6 of the samples, the presence of the anti-IL-10 antibodies promoted complete killing of the parasite. Multiplex analysis of cytokines present within the culture supernatants of the splenic cells revealed a significant increase in both TNF-alpha and IFNg levels in the anti-IL-10 treated cultures, suggesting IL-10 suppression of T cell effector function as one mechanism to account for compromised immunity in these patients. These clinical findings, along with those in experimental models showing that anti-IL-10 receptor antibody promotes rapid control of L. donovani infection and dramatically enhances the leishmanicidal activity of pentavalent antimony, clearly demonstrate the potential benefits of IL-10 inhibition as an approach to therapy in VL.

尽管非活疫苗在实验模型中取得了令人鼓舞的结果，包括基于 DNA 和重组蛋白的疫苗，但安全的非活疫苗在人体试验中始终未能对皮肤或内脏利什曼病提供显着的保护作用。 虽然对这些差异结果可能有多种解释，但实验室试验和现场试验之间的一个关键区别是，与人体试验中的自然白蛉挑战相比，使用针头挑战来评估动物疫苗。 值得注意的是，还没有在受控实验室环境中通过白蛉攻击对实验疫苗进行评估。除了将感染期寄生虫输送到真皮中外，受感染的白蛉还会沉积具有药理活性的唾液和寄生虫衍生的分泌糖蛋白，已知当通过针头与寄生虫共同施用时，每种糖蛋白都会增强疾病的严重程度。 此外，我们最近报道说，白蛉传播会在叮咬部位诱发一种性质独特的炎症反应，其中包括中性粒细胞的动态募集，并且这些中性粒细胞显着增强寄生虫建立原发感染的能力。 我们已经提供了一组明确的数据，表明能够提供针对针刺攻击的强大保护的非活疫苗对白蛉攻击无效。这种差异不是由各自接种物中感染期寄生虫的质量或剂量造成的。 白蛉攻击后中性粒细胞的消耗挽救了灭活疫苗提供保护的能力。传播后 2 周的分析显示，中性粒细胞耗竭导致 CD4+ T 细胞产生 Ag 特异性干扰素 γ (IFNg) 增加。 这些发现表明，咬伤部位早期大量中性粒细胞的募集和持续存在严重损害了皮肤二次免疫的表达。 由于中性粒细胞是利什曼原虫叮咬传播后第一种感染利什曼原虫的细胞类型，并且它们的特异性消耗增强了宿主对利什曼原虫的抵抗力，因此已探索了受感染的中性粒细胞可能遇到未成熟树突状细胞 (iDC) 并损害其功能的可能性。 以下 i.d.将表达 RFP 的 L. Major 接种到溶菌酶-GFP 小鼠中，将回收的 GFP+ 中性粒细胞分为未感染和感染群体，并与 iDC 共培养过夜。 在对 GFP+ 和 GFP- iDC 进行额外分类之后，我们通过在可溶性 OVA 存在下与 OT-I CD8+ T 细胞共培养来研究这些不同 DC 群体的引发能力。 感染的中性粒细胞的摄取抑制了 DC 的成熟，并完全消除了它们刺激 OT-I 细胞增殖和释放 IFNg 的能力。它们的功能障碍比吸收未感染的中性粒细胞的 DC 更加明显，这一差异与受感染的中性粒细胞凋亡状态的增强相关（通过膜联蛋白染色确定）。在体内，中性粒细胞的消耗增加了过继转移的OT-I细胞的增殖，以响应表达OVA的大型利斯特氏菌转基因寄生虫的感染。这些数据证明了利什曼原虫寄生虫诱导免疫失调的新机制，以及受感染的中性粒细胞在对利什曼原虫的免疫反应过程中以前未被认识到的作用。 内源性 IL-10 是免疫稳态的核心介质，对于控制局部组织中伴随抗微生物免疫表达的强烈炎症反应是必要的。 宿主免疫和病理学之间达到平衡的结果可能是慢性或持续性感染，并且 IL-10 的缺失已被证明可以更有效地清除多种病原体，包括硕大利斯特菌。我们最近报道了传统耐药性 C57BL/6 小鼠无法成功解决硕大利斯特菌分离株的感染，尽管宿主有强烈的 IFNg 反应。 易感性是由来自同时产生 IFNg 的 CD4+ 细胞的抗原特异性 IL-10 引起的。在后续研究中，我们探讨了 IL-27 在调节利什曼病 Th1 细胞 IL-10 中的作用。研究发现，IL-27 在体外可增强 L.major 引发的定型 Th1 细胞中 IL-10 的诱导。 对感染 IL-27R 缺陷 (WSX-1-/-) 小鼠病灶和引流淋巴结中 CD4+ 细胞的细胞因子分析显示，IFNg+ CD4+ 细胞中的 IL-10 减少，同时 IFNg+ CD4+ 细胞总数减少，IL-4 增加。 尽管 CD4+ 细胞抑制了 IL-10，但由于 IL-4 失调和 IL-10 残留水平，未观察到寄生虫数量的显着变化。引人注目的是，受感染的 WSX-1-/- 小鼠出现了与 IL-17+ CD4+ 细胞出现相关的严重病变，这表明 IL-27 在硕大利斯特菌感染期间具有阻止不适当 Th17 细胞发育的功能。结果证明了 IL-27 对 L.major 驱动的 Th1、Th2 和 Th17 发育具有多效性作用，并增强了其作为控制免疫与病理之间平衡的关键调节细胞因子的功能。 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。作为控制持续感染引起的炎症的一种可能的稳态机制，抑制性细胞因子 IL-10 在患有活动性疾病的 VL 患者的脾脏、骨髓、PBMC 和血浆中反复观察到水平升高。 在寄生的脾脏中，IL-10 mRNA 升高的来源是 CD3+CD25-Foxp3- 细胞。 直接证明 IL-10 会损害抗原特异性 IFNg 反应并促进靶器官（如脾脏）中的寄生虫生长，这与 VL 的发病机制密切相关。我们利用活检脾细胞内受感染巨噬细胞的存在作为 IL-10 功能的读数。 将用于诊断而获得的少量残余脾抽吸物分成两等体积，并在抗IL-10抗体或同种型对照Ig存在下培养。 3天后，通过在血琼脂平板上连续稀释来定量活寄生虫的数量。 在寄生虫可以生长出来的 18 个配对样品中，14 个样品在与抗 IL-10 孵育 3 天后寄生虫数量减少，而在其中 6 个样品中，抗 IL-10 抗体的存在促进了寄生虫的完全杀死。 对脾细胞培养物上清液中存在的细胞因子的多重分析显示，抗 IL-10 处理的培养物中 TNF-α 和 IFNg 水平显着增加，表明 IL-10 对 T 细胞效应功能的抑制是这些患者免疫力受损的一种机制。 这些临床发现以及实验模型中显示的抗 IL-10 受体抗体可促进杜氏利什曼氏菌感染的快速控制并显着增强五价锑的杀利什曼活性，清楚地证明了 IL-10 抑制作为 VL 治疗方法的潜在益处。

项目成果

Role for CD4(+) CD25(+) regulatory T cells in reactivation of persistent leishmaniasis and control of concomitant immunity.

CD4（+）CD25（+）调节性T细胞在持久性利什曼病的重新激活和对伴随免疫力的控制中的作用。

• DOI: 10.1084/jem.20040298
• 发表时间: 2004-07-19
• 期刊: JOURNAL OF EXPERIMENTAL MEDICINE
• 影响因子: 15.3
• 作者: Mendez, S;Reckling, SK;Piccirillo, CA;Sacks, D;Belkaid, Y
• 通讯作者: Belkaid, Y

Skin dendritic cells in murine cutaneous leishmaniasis.

小鼠皮肤利什曼病的皮肤树突状细胞。

• DOI: 10.1078/0171-2985-00096
• 发表时间: 2001
• 期刊: Immunobiology
• 影响因子: 2.8
• 作者: Udey,MC;vonStebut,E;Mendez,S;Sacks,DL;Belkaid,Y
• 通讯作者: Belkaid,Y

Immunological and pathological evaluation of rhesus macaques infected with Leishmania major.

感染重大利什曼原虫的恒河猴的免疫学和病理学评估。

• DOI: 10.1016/s0014-4894(03)00099-7
• 发表时间: 2003
• 期刊: Experimental parasitology
• 影响因子: 2.1
• 作者: Freidag,BrendaL;Mendez,Susana;Cheever,AllenW;Kenney,RichardT;Flynn,Barbara;Sacks,DavidL;Seder,RobertA
• 通讯作者: Seder,RobertA

Alum-precipitated autoclaved Leishmania major plus bacille Calmette-Guerrin, a candidate vaccine for visceral leishmaniasis: safety, skin-delayed type hypersensitivity response and dose finding in healthy volunteers.

明矾沉淀高压灭菌的重大利什曼原虫加卡介苗，一种内脏利什曼病候选疫苗：健康志愿者的安全性、皮肤迟发型超敏反应和剂量发现。

• DOI: 10.1016/s0035-9203(03)90171-4
• 发表时间: 2003
• 期刊: Transactions of the Royal Society of Tropical Medicine and Hygiene
• 影响因子: 2.2
• 作者: Kamil,AA;Khalil,EAG;Musa,AM;Modabber,F;Mukhtar,MM;Ibrahim,ME;Zijlstra,EE;Sacks,D;Smith,PG;Zicker,F;El-Hassan,AM
• 通讯作者: El-Hassan,AM

Memory may not need reminding.

记忆可能不需要提醒。

• DOI: 10.1038/nm1004-1045
• 发表时间: 2004
• 期刊: Nature medicine
• 影响因子: 82.9
• 作者: Seder,RobertA;Sacks,DavidL
• 通讯作者: Sacks,DavidL