Improving TB vaccine design by natural selection of intracellularly processed epitopes presented in primary tissues of murine model for protective imm

结核分枝杆菌（MTB）发病机制的主要特征之一是它能够通过改变其抗原的表达来操纵免疫应答的时机，以避免早期识别和破坏。结核分枝杆菌具有广泛的组织感染能力[Fanning a， 1999]，肺外结核约占新报告病例的15%和复发病例的20-30%[2017年全球结核病报告，世卫组织]，虽然巨噬细胞是其主要的主要宿主，但其他细胞类型，如专业吞噬细胞（即树突状细胞）和非专业抗原呈递细胞（APCs），即上皮细胞，对病原体是隶属的[Mvubu NE 2016， Harrif MJ, 2014]。Lerner TR 2016]并能加工和呈递MTB抗原[Flyer DC 2002， Penn BH 2018]。宿主对外来抗原的交叉呈递是这一过程的关键部分，在结核分枝杆菌感染中，这主要涉及MHC-I受体与通过细胞内蛋白酶体聚焦加工途径衍生的短肽相关[Adiko AC, 2015；陈华，2016；李文杰，2014]。了解并能够影响这一系统是下一代疫苗设计的一个基本部分。主要的项目假设是，刺激结核分枝杆菌抗原交叉呈递途径可能转化为鉴定下一代疫苗的新的保护性抗原。AIMSObjective 1。目的：探讨卡介苗和结核分枝杆菌在小鼠各器官体外感染后的表现：原发性和继发性淋巴器官泌尿系统呼吸系统心脏系统分化骨髓细胞为了确定卡介苗和结核分枝杆菌在交叉呈递途径上呈递的抗原变化刺激方法和技术-体外感染试验和组织培养的器官收集-免疫原性复合物的提取和纯化-通过质谱法鉴定抗原(s)（协同工作）未来展望任何新鉴定的抗原/方法可以增强其呈递，以提高其有效性，可纳入当前的结核病疫苗接种策略参考文献1。​https://www.who.int/tb/publications/global_report/gtbr2017_main_text.pdf 2。Adiko AC等。MHC I的细胞内转运途径及其与抗原交叉表达的相关性，Front Immunol. 2015年7月2日；6:335。doi: 10.3389 / fimmu.2015.00335。eCollection 2015。第2页。6.范宁。肺外疾病;协会。1999年6月1日；160(11):1597-603；pmi: 10374005Flyer DC等。结核分枝杆菌CD8(+)- t细胞抗原表位的质谱分析[j] .感染免疫学报；2002;20(6):29632。5. hariff MJ等。《公共科学图书馆·综合》，2014年5月14日；9(5):e97515。人类肺上皮细胞在晚期内体液泡中含有结核分枝杆菌，CD8 T细胞有效识别。doi: 10.1371 / journal.pone.0097515。eCollection 2014。6. [J] .结核分枝杆菌抗原表位的功能和潜力[J] .免疫学杂志。2014年3月24日；5:107。doi: 10.3389 / fimmu.2014.00107。eCollection 2014。7. Keller Ch等人。《感染免疫》，2006年7月；74(7):4295-309。8. Lerner等。结核分枝杆菌在淋巴内皮细胞中的复制生态位[J] .中华临床医学杂志，2016;26(3):1093-108。doi: 10.1172 / JCI83379。Epub 2016年2月22日9. Mvubu NE等。“结核分枝杆菌在肺上皮细胞中表现出差异和菌株特异性的分子特征”，Dev Comp Immunol. 2016;65:321329。doi: 10.1016 / j.dci.2016.07.022。Epub 2016 8月3日10. Penn BH等。mmb - human protein - Interaction Map鉴定宿主抗病毒和抗菌反应之间的开关[j] . Mol Cell. 2018, Aug 16;71(4):637-648.e5。

SUMMARYOne of the main hallmarks of Mycobacterium tuberculosis (MTB) pathogenesis is its ability to manipulate the timing of the immune response to its own advantage by changing expression of its antigens to avoid early recognition and destruction. MTB has capacity for infection of a wide range of tissues [Fanning A, 1999] with extrapulmonary TB representing approximately 15% of newly reported cases and 20-30% of relapsed cases [Global Tuberculosis Report 2017, WHO] and while macrophages constitute its main primary host, other cell types, such as professional phagocytes (i.e. dendritic cells) and non-professional antigen presenting cells (APCs) i.e. epithelial cells are submissive to the pathogen [Mvubu NE 2016, Harrif MJ, 2014, Lerner TR 2016] and can process and present MTB antigens [Flyer DC 2002, Penn BH 2018]. Cross-presentation of foreign antigens by host is a critical part of the process and in MTB infection this predominantly involves MHC-I receptors associated with short peptides derived via the intracellular proteasome-focused processing pathway [Adiko AC, 2015; Chen H, 2016; Ivanyi J, 2014]. Understanding and being able to influence this system is a fundamental part of next generation vaccine design. HYPOTHESISThe leading project hypothesis is that stimulation of Mtb antigens cross-presentation pathways may translate in identification of new protective antigens for next generation of vaccines. AIMSObjective 1. To examine BCG and MTB presentation upon in vitro infection in various mouse organs grouped as follows: Primary and secondary lymphoid organs Urinary system Respiratory system Cardiac system Differentiated bone marrow cells Objective 2. To ascertain antigenic changes in BCG and MTB presentation upon cross-presentation pathways stimulation METHODS AND TECHNIQUES- organ collection for in vitro infection assays and tissue culture - extraction and purification of immunogenic complexes - antigen(s) identification via mass spectrometry (collaborative work) FUTURE PROSPECTSAny new identified antigens / methods enhancing their presentation may be included into the current TB vaccination strategies to improve their efficacy REFERENCES1. https://www.who.int/tb/publications/global_report/gtbr2017_main_text.pdf 2. Adiko AC et al. 'Intracellular transport routes for MHC I and their relevance for antigen crosspresentation' Front Immunol. 2015 Jul 2;6:335. doi: 10.3389/fimmu.2015.00335. eCollection 2015. Page 1 of 2 3. Fanning A. Tuberculosis: 6. Extrapulmonary disease; CMAJ. 1999 Jun 1;160(11):1597-603; PMID: 10374005 4. Flyer DC et al. Identification by mass spectrometry of CD8(+)-T-cell Mycobacterium tuberculosis epitopes within the Rv0341 gene product' Infect Immun. 2002 Jun;70(6):292632. 5. Harriff MJ et al. 'Human lung epithelial cells contain Mycobacterium tuberculosis in a late endosomal vacuole and are efficiently recognized by CD8 T cells' PLoS One. 2014 May 14;9(5):e97515. doi: 10.1371/journal.pone.0097515. eCollection 2014. 6. Ivanyj J 'Function and potentials of M.tuberculosis epitopes' Front Immunol. 2014 Mar 24;5:107. doi: 10.3389/fimmu.2014.00107. eCollection 2014. 7. Keller Ch et al. 'Genetically determined susceptibility to tuberculosis in mice causally involves accelerated and enhanced recruitment of granulocytes' Infect Immun. 2006 Jul;74(7):4295-309. 8. Lerner TR et al. Lymphatic endothelial cells are a replicative niche for Mycobacterium tuberculosis J Clin Invest. 2016 Mar 1;126(3):1093-108. doi: 10.1172/JCI83379. Epub 2016 Feb 22. 9. Mvubu NE et al. 'Mycobacterium tuberculosis strains exhibit differential and strain-specific molecular signatures in pulmonary epithelial cells' Dev Comp Immunol. 2016 Dec;65:321329. doi: 10.1016/j.dci.2016.07.022. Epub 2016 Aug 3. 10. Penn BH et al. 'An Mtb-Human Protein-Protein Interaction Map Identifies a Switch between Host Antiviral and Antibacterial Responses' Mol Cell. 2018 Aug 16;71(4):637-648.e5.