背景：

呼吸道感染，特别是下呼吸道感染仍是全球十大死亡原因的第四位。造成呼吸道感染的病原体复杂，包括细菌、真菌、病毒及混合感染，其中最为严重的是耐药菌感染，全世界每年有70万人死于耐药菌感染。目前最严重的耐药细菌是肺炎克雷伯菌、鲍曼不动杆菌、铜绿假单胞菌、肠杆菌、金黄色葡萄球菌和粪肠球菌，这些细菌被称为“ESKAPE”，严重威胁人类健康。但不幸的是新的抗生素研发周期非常缓慢，已无法赶上耐药细菌的发展速度。细菌疫苗是抵抗耐药细菌感染的有效手段，可以大大降低抗生素的使用量，减少细菌耐药性的发展速度。细菌胞外囊泡（EV）由于含有丰富的完整抗原，不具有感染性，易于通过基因工程修饰，并且具有纳米结构，因此它们目前被认为是细菌疫苗的理想成分。然而，其产量和毒性问题一直难以解决。因此，提高囊泡的产量、提高其安全性、增加保护性抗原的覆盖率以及增强其诱导的特异性体液免疫和细胞免疫是改进细菌疫苗设计的关键问题。同时，近年来的研究发现，先天免疫也能够产生免疫记忆，它是指在先天免疫细胞接触到初级刺激后，当识别次级同源或异源刺激物后会激活更强烈的先天免疫应答，这一现象也被称为“训练免疫”。因此，在针对呼吸道感染的防治时，通过诱导训练免疫建立起一种针对多种病原体感染的广谱保护具有十分重要的意义。

目的：

本课题基于高压均质技术可以驱动几乎所有类型的细菌组装成为纳米细菌仿生囊泡（BBV），旨在探讨BBV作为一种安全高产、能够诱导针对耐药细菌体液和细胞免疫双重应答的通用细菌疫苗平台的潜力，从而克服EV作为细菌疫苗的局限性。同时，结合训练免疫能够产生非特异性的免疫应答反应，在BBV细菌疫苗平台的基础上，引入卡介苗细胞壁骨架（BCG-CWS）和酵母细胞壁骨架（Yeast-CWS），开发一种训练免疫诱导剂组合BBVs-CWS，通过其诱导训练免疫建立起一种针对多种病原体感染的广谱保护，为非特异性的预防和治疗呼吸道感染提供了的新的思路。

方法：

（1）使用高压均质技术驱动6种“ESKAPE”细菌自组装为纳米化的细菌仿生囊泡BBV，通过透射电镜、粒度仪、iTRAQ蛋白质组学、SDS-PAGE电泳、核酸电泳等分析BBV囊泡的理化特征。

（2）通过皮下注射荧光标记的BBV观察在注射部位的驻留时间，检测BBV免疫后促进淋巴结树突状细胞（DC）的增殖与成熟，在体外检测小鼠骨髓来源树突状细胞（BMDCs）摄取BBV并促进其活化的情况。

（3）检测BBV免疫小鼠后抗血清识别不同来源细菌的交叉保护性，检测血清中IgG抗体的应答水平以及IgG1与IgG2a抗体的比值。分离BBV免疫后小鼠脾脏淋巴细胞，通过细胞增殖实验和流式细胞分析检测细菌特异性刺激后T细胞的增殖及活化能力。

（4）BBV免疫小鼠后考察其帮助小鼠抵抗细菌引起的脓毒症和急性肺炎的保护效果，评价肺炎模型中BBV疫苗改善炎症不良反应的情况。

（5）制备了另外三种多药耐药的革兰氏阴性菌（鲍曼不动杆菌、铜绿假单胞菌及肠杆菌）的BBV，以及两种多药耐药革兰氏阳性菌（金黄色葡萄球菌、粪肠球菌）的BBV，检测了这些BBV免疫小鼠后特异性抗体水平和细菌攻毒后的保护效果。

（6）监测BBV免疫期间小鼠的体重与体温变化，对免疫后小鼠器官组织进行H&E切片染色，观察组织病变和炎性细胞浸润。

（7）BBVs+CWS在体外训练小鼠骨髓来源的巨噬细胞（BMDMs），随后使用异源细菌进行挑战，检测细胞因子、乳酸、乙酰辅酶A生成水平，考察其促进BMDMs对异源细菌吞噬和胞内杀伤能力的提升。

（8）考察糖酵解抑制剂（2-DG）和氧化磷酸化抑制剂(Oligomycin)对BBVs+CWS在体外训练BMDMs细胞因子产生、细菌吞噬和胞内杀伤的影响。

（9）BBVs+CWS诱导小鼠呼吸道训练免疫后对异源鲍曼不动杆菌挑战的保护作用。

（10）转录组学分析小鼠肺部在BBVs+CWS训练前后的功能差异。

（11）考察BBVs+CWS诱导小鼠呼吸道训练免疫后对白色念珠菌全身感染的保护效果。以及对流感病毒呼吸道感染的保护效果和特异性抗体的产生水平。

结果：

（1）利用高压均质技术驱动6种“ESKAPE”细菌挤过夹缝形成出芽水泡，释放了胞内蛋白及核酸后，由细菌细胞膜自组装形成囊泡。相比于EV，BBV产量更高并保留了完整的膜抗原。

（2）BBV能够在注射部位长时间驻留达120h，BBV能够快速进入淋巴结并被DCs细胞高效摄取和加工，在体内和体外都能够高效促进DCs细胞的活化和成熟。

（3）BBV小鼠免疫后的抗血清可以识别三种不同来源的耐药肺炎克雷伯菌，并产生了比EV更强的细菌特异性IgG抗体应答，BBV作为疫苗对机体产生了偏向Th1型的免疫应答能力。

（4）BBV疫苗免疫后分离的脾脏淋巴细胞具有针对不同来源的灭活耐药肺炎克雷伯菌的刺激有最显著的增殖能力和细胞因子生成水平，BBV疫苗能够诱导最强的CD3⁺T细胞的细菌特异性应答，且对不同来源的细菌具有交叉反应能力。

（5）经BBV免疫小鼠后，可以显著提高小鼠遭到脓毒症挑战时的生存率，降低急性细菌性肺炎感染后的细菌载量和炎症反应，同时在肺部募集了大量CD4⁺T和CD8⁺T细胞。

（6）高压均质制备技术不仅可以驱动多种革兰氏阴性菌产生BBV，也能驱动厚细胞壁的革兰氏阳性菌细菌形成BBV，由革兰氏阳性菌形成的BBV也可以作为疫苗诱导有效的体液及细胞免疫应答。

（7）通过对免疫程序过程中小鼠体重和体温的监测，以及不同组织器官H&E分析显示2μg的BBV皮下免疫并不会对小鼠造成明显的毒性和组织损伤。

（8）BBVs+CWS在体外训练BMDMs后，在异源细菌挑战时产生了最强烈的炎性细胞因子TNF-α、IL-6和趋化因子CXCL2的释放，以及最明显的乳酸和乙酰辅酶A含量的增加，同时展现出最强的异源细菌的吞噬能力和最高效的细菌胞内杀伤率。

（9）糖酵解抑制剂和氧化磷酸化抑制剂显著抑制了BBVs+CWS在体外训练BMDMs后细胞因子产生水平、细菌吞噬和胞内杀伤能力。

（10）BBVs+CWS诱导小鼠呼吸道训练免疫后能够快速清除异源细菌感染，并降低肺部炎症水平，并在训练后60天依然具有保护效果。

（11）肺组织的RNA-seq分析显示BBVs-CWS诱导的训练免疫增强了某些特定炎症基因的表达，促进了关键转录因子的活化，促进氧化磷酸化向有氧糖酵解过程的转变。

（12）BBVs+CWS的呼吸道训练免疫可以有效保护小鼠抵抗白色念珠菌的全身性感染，并显著降低了流感病毒肺部感染时的病毒载量，产生了最高水平的病毒特异性IgG、IgA抗体，并诱导了Th1型偏向的抗体应答。

结论：

在本论文第一部分的研究中，我们创新性的开发了一种利用超高压强驱动细菌稳定形成细菌仿生囊泡（BBV）的技术平台，该技术可以驱动包括革兰氏阴性菌及革兰氏阳性菌在内的六种简称为“ESKAPE”的耐药细菌产生各自的BBV。这种BBV释放了细菌的胞内蛋白及核酸，相比细菌天然分泌的EV，具有更高的产量及安全性。BBV可以高效的激活DC细胞，具有诱导细菌特异性的体液及细胞免疫应答的双重功能。BBV作为疫苗对革兰氏阴性和阳性细菌感染都有显著的保护作用，具有成为通用细菌疫苗技术平台的潜力。在本论文第二部分的研究中，结合训练免疫能够产生非特异性广谱的免疫应答反应，在前期BBV细菌疫苗平台的基础上，引入卡介苗细胞壁骨架（BCG-CWS）和酵母细胞壁骨架（Yeast-CWS），首次开发了一种训练免疫诱导剂组合BBVs-CWS。BBVs-CWS在体外能够诱导BMDMs细胞训练免疫并在异源细菌挑战时产生更强烈的炎症应答，促进细胞对细菌的吞噬和胞内杀伤能力。在体内，BBVs-CWS诱导的呼吸道训练免疫能够快速清除异源细菌感染，并降低肺部炎症水平，并在训练后60天依然具有保护效果。同时BBVs-CWS诱导的呼吸道训练免疫对全身性真菌感染和肺部病毒感染依然具有显著的保护作用，因此BBVs+CWS具有作为一种广谱的训练免疫诱导剂的重要潜力。

Background: 

Respiratory infections, especially lower respiratory tract infections, remain the fourth among the top 10 causes of death worldwide. The pathogens causing respiratory tract infections are complex, including bacterial, fungal, virus and mixed infections. The most serious of them is drug-resistant infections, which kills 700,000 people worldwide every year. At present, the most serious drug-resistant bacteria are Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter, Staphylococcus aureus and Enterococcus faecalis, which are called "ESKAPE" and pose a serious threat to human health. Unfortunately, the development cycle of new antibiotics is too slow to keep up with the development of resistant bacteria. Bacterial vaccines are an effective means of combating drug-resistant bacterial infections, which can greatly reduce the use of antibiotics and the development of bacterial resistance. Bacterial extracellular vesicles (EV) are currently considered as ideal components of bacterial vaccines because they contain abundant intact antigens, are non-infectious, can be easily modified by genetic engineering, and have nanostructures. However, the issues of yield and toxicity have been difficult to resolve. Therefore, increasing the yield of vesicles, improving their safety, increasing the coverage of protective antigens, and enhancing the specific humoral and cellular immunity induced by them are key issues to improve the design of bacterial vaccines. At the same time, recent studies have found that innate immunity can also produce immune memory, which means that after the innate immune cells are exposed to the primary stimulus, they will activate a stronger innate immune response when they recognize the secondary homologous or heterologous stimulus, which is also called "training immunity". Therefore, in the prevention and treatment of respiratory tract infections, it is of great significance to establish a broad spectrum of protection against a variety of pathogens by inducing training immunity.

Objective: 

Based on the high-pressure homogenization technology, this project can drive the assembly of almost all types of bacteria into nano-bacterial bionic vesicles (BBV), aiming to explore the potential of BBV as a safe and high-yield universal bacterial vaccine platform that can induce dual humoral and cellular immune responses against drug-resistant bacteria, thereby overcoming the limitations of EV as bacterial vaccines. At the same time, combined with training immunity, nonspecific immune responses can be produced. On the basis of BBV bacterial vaccine platform, a combination of training immune inducer BBVs-CWS was developed by introducing BCG cell wall skeleton (BCG-CWS) and Yeast cell wall skeleton (Yeast-CWS). Through the induction of training immunity, a broad-spectrum protection against a variety of pathogens is established, which provides a new idea for the non-specific prevention and treatment of respiratory tract infection.

Methods:

(1) Six "ESKAPE" bacteria were driven to self-assemble into nano-sized bacterial bionic vesicles BBV by high-pressure homogenization technology. The physicochemical characteristics of BBV vesicles were analyzed by transmission electron microscopy, particle size analyzer, iTRAQ proteomics, SDS-PAGE electrophoresis, and nucleic acid electrophoresis.

(2) The residence time of fluorescently labeled BBV at the injection site was observed, the proliferation and maturation of dendritic cells (DC) in lymph nodes were detected after BBV immunization, and the uptake and activation of bone marrow-derived dendritic cells (BMDCs) in vitro were detected.

(3) The cross-protection of BBV antiserum against bacteria from different sources was detected by detecting the level of IgG antibody response and the ratio of IgG1 to IgG2a antibody. Spleen lymphocytes were isolated from mice immunized with BBV. The proliferation and activation of T cells after specific stimulation were detected by cell proliferation assay and flow cytometry analysis.

(4) To evaluate the protective effect of BBV vaccine against bacterial sepsis and acute pneumonia in mice, and to evaluate the improvement of inflammatory side effects in the pneumonia model.

(5) BBV against three other multidrug resistant Gram-negative bacteria (Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter) and two multidrug resistant Gram-positive bacteria (Staphylococcus aureus and Enterococcus faecalis) were prepared. The levels of specific antibodies and the protective effects of these BBV against bacterial challenge in mice were detected. 

(6) The changes of body weight and body temperature of mice during BBV immunization were monitored, and the organs and tissues of mice after immunization were stained with H&E to observe tissue lesions and inflammatory cell infiltration.

(7) BBVs+CWS was used to train mouse bone marrow-derived macrophages (BMDMs) in vitro, and then challenged with heterologous bacteria. The levels of cytokines, lactate, and acetyl-coa were detected to investigate the enhancement of BMDMs' phagocytosis and intracellular killing ability against heterologous bacteria.

(8) The effects of glycolysis inhibitor and oxidative phosphorylation inhibitor on cytokine production, bacterial phagocytosis and intracellular killing of BMDMs cultured with BBVs+CWS in vitro were investigated.

(9) BBVs+CWS induced protection against heterologous Acinetobacter baumannii challenge in mice after respiratory training.

(10) Transcriptome analysis of functional differences in mouse lungs before and after BBVs+CWS training.

(11) To investigate the protective effect of BBVs+CWS on systemic Candida albicans infection in mice after respiratory training. And the protective effect against influenza virus respiratory tract infection and the level of specific antibody production.

Results:

(1) Six strains of "ESKAPE" bacteria were driven through the cracks to form budding vesicles by high-pressure homogenization technology. After the release of intracellular proteins and nucleic acids, the bacterial cell membrane self-assembled to form vesicles. Compared with EV, BBV produced higher yield and retained intact membrane antigens.

(2) BBV can stay in the injection site for a long time up to 120 hours, and can quickly enter the lymph nodes and be efficiently taken up and processed by DCs. BBV can effectively promote the activation and maturation of DCs both in vivo and in vitro.

(3) The antiserum of BBV immunized mice could recognize three kinds of drug-resistant Klebsiella pneumoniae strains from different sources and produced stronger bacteria-specific IgG antibody responses than EV, suggesting that BBV as a vaccine produced Th1-biased immune responses.

(4) Spleen lymphocytes isolated after BBV vaccination had the most significant proliferation ability and cytokine production in response to inactivated and drug-resistant Klebsiella pneumoniae from different sources. BBV vaccine could induce the strongest bacteria-specific response of CD3⁺T cells, and had the ability to cross-react with different bacteria.

(5) BBV could significantly improve the survival rate of mice challenged with sepsis, reduce bacterial load and inflammatory response after acute bacterial pneumonia infection, and recruit a large number of CD4⁺ and CD8⁺T cells in the lung.

(6) High-pressure homogenization preparation technology can not only drive a variety of Gram-negative bacteria to produce BBV, but also drive thick cell wall Gram-positive bacteria to produce BBV. BBV produced by Gram-positive bacteria can also be used as a vaccine to induce effective humoral and cellular immune responses.

(7) By monitoring the body weight and body temperature of mice during the immunization program, and H&E analysis of different tissues and organs, it was shown that 2μg BBV subcutaneous immunization did not cause obvious toxicity and tissue damage to mice.

(8) After in vitro training BMDMs with BBVs+CWS, the strongest release of inflammatory cytokines TNF-α, IL-6 and chemokine CXCL2, the most obvious increase of lactate and acetyl-coa content, the strongest phagocytosis ability of heterologous bacteria and the most efficient bacterial intracellular killing rate were observed when challenged with heterologous bacteria.

(9) The glycolysis inhibitor (2-DG) and oxidative phosphorylation inhibitor (Oligomycin) significantly inhibited the cytokine production level, bacterial phagocytosis and intracellular killing ability of BMDMs cultured with BBVs+CWS in vitro.

(10) BBVs+CWS could rapidly clear heterologous bacterial infection and reduce the level of pulmonary inflammation in mice after respiratory training, and it still had protective effect 60 days after training.

(11) RNA-seq analysis of lung tissues revealed that BBVs-CWS induced trained immunity enhanced the expression of specific inflammatory genes, promoted the activation of key transcription factors, and promoted the transition of oxidative phosphorylation to aerobic glycolysis.

(12) BBVs+CWS could effectively protect mice against systemic infection with Candida albicans, significantly reduce the viral load during pulmonary infection with influenza virus, produce the highest level of virus-specific IgG and IgA antibodies, and induce Th1-biased antibody responses.

Conclusions:

In the first part of this thesis, we developed a novel bacterial biomimetic vesicle (BBV) platform using ultra-high pressure to drive the production of BBV in six drug-resistant bacterial species, including Gram-negative and Gram-positive bacteria, referred to as "ESKAPE". This BBV releases bacterial intracellular proteins and nucleic acids, and has higher production and safety compared with EV naturally secreted by bacteria. BBV can efficiently activate DC cells and induce both humoral and cellular immune responses to bacteria. As a vaccine, BBV has a significant protective effect against both Gram-negative and positive bacterial infections and has the potential to become a universal bacterial vaccine technology platform. In the second part of this thesis, a combination of trained immune inducers (BBVs-CWS) was developed based on the previous BBV bacterial vaccine platform by introducing Bacillus Calmette-Guerin cell wall skeleton (BCG-CWS) and Yeast cell wall skeleton (Yeast-CWS). BBVs-CWS can induce BMDMs cell-trained immunity and produce stronger inflammatory responses when challenged with heterologous bacteria in vitro, and promote the phagocytosis and intracellular killing ability of BMDMs. In vivo, BBVs-CWS induced respiratory training immunity could rapidly clear heterologous bacterial infection and reduce the level of lung inflammation, and the protective effect was still maintained up to 60 days after training. At the same time, the respiratory training immunity induced by BBVs-CWS still has a significant protective effect on systemic fungal infection and pulmonary viral infection. Therefore, BBVs+CWS has the important potential as a broad-spectrum training immunity inducer.