Immuno dominant Mycobacterium tuberculosis Protein Rv1507A Elicits Th1 Response and Modulates Host Macrophage Effector Functions

结核分枝杆菌免疫优势蛋白Rv1507A激发Th1反应并调控宿主巨噬细胞效应功能

INTRODUCTION

引言

Mycobacterium tuberculosis (M. tb), the intracellular pathogen causing Tuberculosis (TB), still cause nearly 10 million new TB cases annually and about 1.45 million deaths were reported in 2018 alone (1). It is therefore imperative that prevention strategies such as development of new vaccines against TB be undertaken on priority. Bacillus Calmette Guerin (BCG) vaccine provides a degree of immunity against M. tb infections in children, however its efficacy is variable among adults populations due to its inability to mount a strong and sustained memory response (2). Development of new TB vaccine or improving the efficacy of BCG is therefore important to achieve the goals laid down in END-TB strategy (3). The emerging role of trained immunity and enhanced efficacy of BCG by altering the vaccination route has reinvigorated the interest toward live vaccines (4–7). Recombinant BCG vaccines like rBCG30, BCG expressing RD1 proteins, rBCG 1ureC::hly (VPM1002), etc. have been found to provide enhanced protection as compared to wild type BCG (8, 9). Recombinant M. smegmatis based vaccines have been also shown to induce effective immunity against M. tb with considerable protective potential (10–13). Exploratory studies to identify antigens that can enhance the potency of BCG vaccine are exigently warranted.

结核分枝杆菌（M. tb）作为引发结核病（TB）的胞内病原体，每年仍导致近1000万新增病例，仅2018年就报告约145万死亡病例 (1)。因此必须优先实施预防策略，例如开发新型结核疫苗。卡介苗（BCG）虽能为儿童提供一定免疫保护，但由于其无法产生强效持久的记忆应答，对成人群体保护效果存在差异 (2)。开发新型结核疫苗或提升卡介苗效力，对实现"终止结核病"战略目标至关重要 (3)。训练免疫的新兴作用及通过改变接种途径提升卡介苗效力的研究，重新激发了人们对活疫苗的关注 (4-7)。与野生型卡介苗相比，重组卡介苗如rBCG30、表达RD1蛋白的BCG、rBCG 1ureC::hly（VPM1002）等已被证实能提供更强保护力 (8, 9)。基于重组耻垢分枝杆菌的疫苗也显示出诱导抗结核有效免疫的潜力 (10-13)。当前亟需开展探索性研究以鉴定能增强卡介苗效力的抗原。

M. tb primarily infects and resides within macrophages. The ph ago cyto zed M. tb can escape phago-lysosomal fusion, avoid lysis within macrophage, and modulate the activity of other immune cells by regulating cytokine secretion (14). M. tb also impairs the function of APCs by modulation of antigen presentation or expression of co-sti mula tory molecules (15, 16). Cell-mediated immunity regulated by Th1 cytokines is considered important to eradicate the intracellular pathogens while Th2 response has been found to have important role in clearing the extracellular infectious agents (17). Although some vaccines do suggest a role of Th17 cells in protection particularly during mucosal administration, the exact role is still controversial (18–21). Increased focus on mucosal based vaccines in recent years suggest that IL-17A responses may be necessary for effective vaccination-induced anti-mycobacteria l immunity in the lung (22). Th1 cytokine IFN $\gamma$ helps in activation of macrophages and ph ago lysosome formation that lead to production of reactive oxygen and nitrogen species, resulting in elimination of infection (23). TNF $\alpha$ works in tandem with IFN $\gamma$ and helps in activation of macrophages and in production of reactive oxygen and nitrogen species, thereby mediating the b acte rio static function of macrophages (24, 25). IFN $\gamma$ and TNF $\alpha$ knocked out mice were found to be more susceptible to M. tb infection (26). Therefore, vaccines or protein antigens that boost Th1 immune response will likely be more effective to control M. tb infection, though this is not the only protective signature (27, 28). Moreover, despite evoking Th1 response, some vaccines were nevertheless ineffective, demanding exploratory vaccine studies to predict novel biomarkers of protection (29).

结核分枝杆菌(M. tb)主要感染并定植于巨噬细胞内。被吞噬的M. tb能逃逸溶酶体融合，避免在巨噬细胞内被溶解，并通过调节细胞因子分泌来调控其他免疫细胞活性 [14]。该菌株还会通过调控抗原呈递或共刺激分子表达来削弱抗原呈递细胞(APC)功能 [15, 16]。由Th1细胞因子调控的细胞免疫被认为对清除胞内病原体至关重要，而Th2应答则在清除胞外感染源中发挥重要作用 [17]。虽然部分疫苗（尤其是黏膜接种时）确实提示Th17细胞具有保护作用，但其确切机制仍存争议 [18-21]。近年来对黏膜疫苗的深入研究表明，IL-17A应答可能是肺部疫苗诱导抗结核免疫的必要条件 [22]。Th1细胞因子IFN-γ可激活巨噬细胞并促进吞噬溶酶体形成，进而产生活性氧/氮物质以清除感染 [23]。TNF-α与IFN-γ协同作用，既能激活巨噬细胞，又能促进活性氧/氮物质产生，从而介导巨噬细胞的抑菌功能 [24, 25]。IFN-γ和TNF-α基因敲除小鼠对M. tb感染更易感 [26]。因此，虽然Th1免疫应答并非唯一保护性特征 [27, 28]，但能增强该反应的疫苗或蛋白抗原可能对控制M. tb感染更有效。值得注意的是，某些疫苗虽能激发Th1应答却仍无效，这要求探索性疫苗研究需预测新的保护性生物标志物 [29]。

Studies to decipher the role of genes and their encoded proteins that aid in virulence of $M.$ . tb have been at the center stage of research. Numerous efforts have been made to unravel the immuno modulator y effect of mycobacteria l proteins to gain insights into pathogen es is or development of novel vaccine candidates (30–32). Many of the proteins are known to activate cell mediated Th1 type host immune responses (33–35). Several other proteins have also been unveiled that hamper the immune responses and play a role in pathogen es is (36–40). Numerous attempts have been made to incorporate M. tb proteins into non-pathogens to enhance their vaccine potential (41). Though the strategy is feasible, evidence of reversion to virulence has also been reported (42). Thus, candidate proteins need to be examined, in-vitro as well as in-vivo, for further exploration as a vaccine candidate. We earlier reported a comparative genomic and proteomic analysis of several opportunistic, non-pathogenic and pathogenic species of mycobacteria to show that M. tb evolved from non-pathogenic soil-dwelling bacteria through genome reduction (43). In this process, several key genes that were essential for survival and virulence of M. tb were either retained or expanded (44). Elucidating the function and immunological significance of gene products, present in M. tb but absent in non-pathogenic mycobacteria l species, are therefore important. Taking clue from these studies we identified Rv1507A gene, the so-called “signature protein” present exclusively in M. tb. This gene is present in RD4 region of M. tb affirming its absence in BCG. Our analysis also revealed its exclusive presence in M. tb and absence from all mycobacteria l species studied therein. We describe immunological and other attributes of this otherwise hypothetical protein in terms of its likely utility as a vaccine candidate that demands further exploration.

解析基因及其编码蛋白在结核分枝杆菌($M.$ tb)毒力中作用的研究一直是科学探索的核心领域。为阐明分枝杆菌蛋白的免疫调节效应，学界已开展大量工作以深入理解致病机制或开发新型候选疫苗[30-32]。已知多种蛋白可激活细胞介导的Th1型宿主免疫应答[33-35]，同时也发现另一些蛋白会抑制免疫反应并参与致病过程[36-40]。多项研究尝试将结核分枝杆菌蛋白导入非致病菌以增强疫苗潜力[41]，尽管该策略可行，但已有毒力恢复的报道[42]。因此需通过体外和体内实验验证候选蛋白的疫苗适用性。我们前期通过比较基因组学与蛋白质组学分析发现，结核分枝杆菌由非致病性土壤菌通过基因组精简进化而来[43]，在此过程中保留或扩增了对其生存和毒力至关重要的关键基因[44]。因此阐明结核分枝杆菌特有而非致病性分枝杆菌缺失的基因产物的功能及免疫学意义尤为重要。基于这些研究，我们鉴定出Rv1507A基因——这种仅存在于结核分枝杆菌的"特征蛋白"。该基因位于结核分枝杆菌RD4区，证实了其在卡介苗中的缺失。我们的分析还显示其在所有研究的分枝杆菌物种中具有结核分枝杆菌特异性。本文将阐述这一假设蛋白的免疫学特性及其他特征，探讨其作为需进一步研究的候选疫苗的潜在价值。

MATERIALS AND METHODS

材料与方法

Reagents and Cell Culture

试剂与细胞培养

Mycobacterium smegmatis $\mathrm{m}c^{2}155_{\mathrm{:}}$ obtained from ATCC (Virginia, United States), was maintained as glycerol stocks in our laboratory. Middle brook 7H9 growth media supplemented with $10%$ OADC (Himedia Laboratories, Mumbai, India) was used to subculture bacterial strains. DMEM supplemented with $10%$ Fetal Bovine Serum (FBS), hereafter called complete media, was used for sub culturing RAW264.7 cells. Isopropyl -D-1- thi oga lac to pyr a no side (IPTG), Sarcosyl, imidazole, kanamycin, St a uro spor in e, and MTT were obtained from Merck Limited, Mumbai, India. All cell culture reagents including DMEM were procured from Gibco (Thermo Fisher Scientific India Pvt Ltd, Mumbai, India). Antibodies, Middle brook 7H11 agar, Middle brook 7H9 agar, and 7H10 media were obtained from BD Bioscience s (San Jose, CA, USA). Enzymes, ELISA kit, and toxicity removal kit were obtained from NEB (Massachusetts, USA), PeproTech US (Rocky Hill, NJ, USA), and Norgen (Thorold, ON, Canada), respectively, or otherwise mentioned. All reagents used in our experiments were of analytical grade.

耻垢分枝杆菌 $\mathrm{m}c^{2}155_{\mathrm{:}}$ 购自美国弗吉尼亚州ATCC，以甘油保藏形式保存于本实验室。使用添加了10% OADC (印度孟买Himedia Laboratories) 的Middle brook 7H9培养基传代细菌菌株。采用添加10%胎牛血清(FBS)的DMEM培养基(后称完全培养基)传代RAW264.7细胞。异丙基-D-1-硫代半乳糖苷(IPTG)、十二烷基肌氨酸钠、咪唑、卡那霉素、星形孢菌素及MTT购自印度孟买Merck Limited。包括DMEM在内的所有细胞培养试剂均采购自Gibco (印度孟买Thermo Fisher Scientific India Pvt Ltd)。抗体、Middle brook 7H11琼脂、Middle brook 7H9琼脂及7H10培养基购自美国加州圣何塞BD Biosciences。酶制剂、ELISA试剂盒及毒性去除试剂盒分别购自美国马萨诸塞州NEB、美国新泽西州Rocky Hill的PeproTech US及加拿大安大略省Thorold的Norgen公司(特殊标注除外)。本实验所用试剂均为分析纯级别。

In-silico Structural and Functional Analyses of Rv1507A Protein

Rv1507A蛋白的计算机结构及功能分析

The prediction of protein binding sites in disordered regions was done using Anchor (https://iupred2a.elte.hu) and T cell/B cell epitope prediction was done by IEDB ( tools.immune epi to pe.org/) tool. The subcellular localization and secretory nature of this protein was determined by Predict Protein (https://www.predict protein.org/).

无序区域中蛋白质结合位点的预测使用Anchor (https://iupred2a.elte.hu)完成，T细胞/B细胞表位预测通过IEDB (http://tools.immuneepitope.org/)工具实现。该蛋白质的亚细胞定位和分泌特性由Predict Protein (https://www.predictprotein.org/)确定。

Cloning, Expression, Purification of M. tb Rv1507A, and Generation of M. smegmatis Knock-in

结核分枝杆菌 Rv1507A 的克隆、表达、纯化及耻垢分枝杆菌敲入株的构建

In order to evaluate the secretory nature Rv1507A, the presence of this protein in culture filtrate of M. tb was evaluated by SDS-PAGE analysis. The gel was blotted onto PVDF membrane and probed with polyclonal sera raised against Rv1507A. The ORF encoding M. tb Rv1507A gene was amplified using polymerase chain reaction (PCR) using forward and reverse primers generated from $M.$ . tb $\mathrm{H}_{37}\mathrm{Rv}$ genomic database (Supplementary Table 1). $R\nu I507A$ was inserted in pET28a expression vector (Addgene, Massachusetts, USA) using EcoRI and HindIII restriction sites to construct plasmid pET 28 a Rv 1507 A. E.coli BL21(DE3) expression strain was transformed with recombinant constructs and the culture was incubated with $1\mathrm{mM}$ IPTG for $^{3\mathrm{h}}$ at $37^{\circ}C$ to induce expression of recombinant protein, Rv1507A, which was purified using NiNTA affinity column (Qiagen, Hilden, Germany) and eluted with $300\mathrm{mM}$ imidazole (45). The dialyzed protein was concentrated using centricons (Merck Limited, Mumbai, India) with $3\mathrm{kDa}$ cut off, treated with Polymyxin B beads (Merck Limited, Mumbai, India) to remove bacterial endotoxin contamination and examined using SDS-PAGE.

为了评估Rv1507A的分泌特性，通过SDS-PAGE分析检测了该蛋白在结核分枝杆菌(M. tb)培养滤液中的存在。凝胶被转印至PVDF膜上，并用针对Rv1507A的多克隆血清进行探测。使用从结核分枝杆菌H37Rv基因组数据库(附表1)生成的引物，通过聚合酶链式反应(PCR)扩增编码M. tb Rv1507A基因的开放阅读框(ORF)。Rv1507A被插入到pET28a表达载体(Addgene, Massachusetts, USA)的EcoRI和HindIII限制性酶切位点，构建质粒pET28a-Rv1507A。用重组构建体转化大肠杆菌BL21(DE3)表达菌株，培养物在37°C下用1mM IPTG诱导3小时表达重组蛋白Rv1507A，该蛋白通过NiNTA亲和柱(Qiagen, Hilden, Germany)纯化，并用300mM咪唑洗脱[45]。透析后的蛋白使用3kDa截留分子量的centricons(Merck Limited, Mumbai, India)浓缩，用多粘菌素B珠(Merck Limited, Mumbai, India)处理以去除细菌内毒素污染，并通过SDS-PAGE检测。

Mycobacteria l integration expression vector pST-Ki was used to sub clone Rv1507A gene from pET 28 a Rv 1507 A construct to create pST-Ki_Rv1507A plasmid, as described previously (46). Electro p oration (Bio-Rad laboratories, California, United States) of pST-Ki_Rv1507A into M. smegmatis was carried out to generate M. smegmatis Rv1507A knock-in. Kanamycin $(50\mathrm{g/ml})$ containing Middle brook 7H11 agar plates supplemented with $0.5%$ glycerol were used to select the positive colonies and confirmed through PCR amplification using the standard protocols, as described previously (40). The integration of pST-Ki_Rv1507A into the genome of $M.$ . smegmatis was confirmed by a three step sequential process. Firstly, M. smegmatis positive colonies harboring Rv1507A $(\mathrm{Ms}_\mathrm{Rv}1507\mathrm{A})$ and vector pST-Ki (Ms_Vc) were grown on kanamycin containing 7H11 agar plates. Positive colonies were selected and consecutively passaged for seven generations on antibiotic containing plates. In the second step, the selected colonies were plated on kanamycin negative plate and passaged for five generations. Lastly, the integration of cassette was confirmed by again plating on kanamycin plate and passaged for seven generations. Confirmed colonies were grown till log phase, harvested, centrifuged and the pellet was heated at $95^{\circ}\bar{\mathrm{C}}$ for $30\mathrm{min}$ after dissolving in SDS-PAGE loading dye. Lysate fractions were centrifuged at $13,000~\mathrm{rpm}$ for $10\mathrm{min}$ and the super nat ant was loaded on $10%$ Tricine gel. Rv1507A protein was confirmed by western blotting, using anti-rabbit polyclonal Rv1507A antibody generated in rabbit (described below). The blots were visualized after incubation with HRP labeled anti-rabbit IgG antibody.

按照之前描述的方法[46]，使用分枝杆菌整合表达载体pST-Ki从pET28a_Rv1507A构建体中亚克隆Rv1507A基因，构建pST-Ki_Rv1507A质粒。将pST-Ki_Rv1507A通过电转仪(Bio-Rad laboratories, California, United States)转入耻垢分枝杆菌，构建耻垢分枝杆菌Rv1507A敲入株。采用含卡那霉素$(50\mathrm{g/ml})$的Middlebrook 7H11琼脂平板(添加$0.5%$甘油)筛选阳性克隆，并参照文献[40]标准流程进行PCR验证。

通过三步连续传代法确认pST-Ki_Rv1507A在耻垢分枝杆菌基因组中的整合：首先，将携带Rv1507A的阳性克隆$(\mathrm{Ms}_\mathrm{Rv}1507\mathrm{A})$和空载体pST-Ki对照株(Ms_Vc)接种于含卡那霉素的7H11平板，连续传代7次；第二步，将筛选菌落接种至无抗生素平板传代5次；最后重新接种至含卡那霉素平板传代7次。将验证成功的菌株培养至对数期，离心收集菌体沉淀，溶解于SDS-PAGE上样缓冲液后$95^{\circ}\mathrm{C}$加热$30\mathrm{min}$，$13,000~\mathrm{rpm}$离心$10\mathrm{min}$取上清进行$10%$Tricine凝胶电泳。使用兔源多克隆抗Rv1507A抗体(制备方法见下文)通过Western blot验证蛋白表达，HRP标记的兔IgG二抗孵育后显色观察。

Immunization of Mice

小鼠免疫接种

All experiments using lab animals were conducted as per the guidelines provided by the Committee for the Purpose of Control and Supervision on Experiments on Animals (CPCSEA) (www.envfor.nic.in/divisions/awd/ cpc sea laboratory.pdf) and in compliance with the protocols approved by Institutional Biosafety Committee and Institutional Animal Ethics Committee, National Institute of Pathology, New Delhi, India (Approval No. NIP/IAEC-1701). All animals were housed in positive-pressure units under ambient conditions ${}^{25^{\circ}}{\mathrm{C}},$ $12\mathrm{h}$ light/dark cycle). Purified recombinant protein M. tb Rv1507A $(200\mathrm{g/ml})$ , emulsified with an equal volume of Freund’s incomplete adjuvant (Merck Limited, Mumbai, India), was injected subcutaneous ly in rabbits at two different sites (0.5 ml/site) followed by two booster immunizations $(100\mathrm{g/ml})$ with Freund’s incomplete adjuvant at 15 day intervals. Titer of polyclonal antibodies against M. tb Rv1507A in rabbit sera was determined by dot-blot technique 2 weeks after final immunization.

所有实验动物操作均遵循印度动物实验控制与监督委员会(CPCSEA)(www.envfor.nic.in/divisions/awd/cpcsea_laboratory.pdf)指南，并符合印度新德里国家病理学研究所机构生物安全委员会和动物伦理委员会批准的实验方案(批准号:NIP/IAEC-1701)。所有动物饲养在25℃恒温、12小时光暗循环的正压环境中。将纯化的重组蛋白M. tb Rv1507A(200μg/ml)与等体积弗氏不完全佐剂(Merck Limited，印度孟买)乳化后，于家兔两个不同部位皮下注射(0.5毫升/部位)，随后每隔15天用弗氏不完全佐剂加强免疫两次(100μg/ml)。末次免疫2周后采用斑点印迹法检测家兔血清中抗M. tb Rv1507A多克隆抗体的效价。

Inbred BALB/c mice (Female, 8–12 week, $20\mathrm{-}25\mathrm{g})$ were obtained from the National Institute of Immunology (New Delhi, India). Primary immunization was carried out with purified recombinant M. tb Rv1507A protein $(10~\mathrm{g})$ in $100~\mathrm{l}$ PBS buffer, administered subcutaneous ly. We avoided use of adjuvant to minimize the immuno modulator y bias introduced by use of adjuvants (47, 48). A booster dose of this protein ( $10~\mathrm{g}$ in $100~\mathrm{l}$ PBS) was administered on 10th day of immunization followed by another booster after 10 days. Control animals were injected with equal amount of PBS at the immunizing site. Thirty days after primary immunization, mice were sacrificed to isolate sp leno cyte s for other functional assays. Thereafter, in a separate set of experiments, BALB/c mice (female, $^{8-}$ 12 week, 20–25g) were obtained from National Institute of Biological s (Noida, India) and randomized into three groups $\overset{\cdot}{n}=6/$ group). Mice were intra-peritoneal ly injected with PBS, $\mathrm{Ms_Vc}_{}$ or Ms_Rv1507A ( $\mathrm{1}\times\mathrm{10^{7}}$ CFU), respectively, to evaluate the antigen i city and immunogen i city of this protein (49–51). Groups of mice were also infected intra-tracheally to mimic aerogenic infection and gross pathology of lungs was observed as explained in the following. Animals were randomized and treated similarly as discussed above for intra-peritoneal infections except bacteria were delivered via intra-tracheal in still ation directly to lung surface.

近交系BALB/c小鼠(雌性, 8-12周龄, $20\mathrm{-}25\mathrm{g}$)购自印度新德里国家免疫学研究所。初次免疫采用纯化重组结核分枝杆菌Rv1507A蛋白$(10~\mathrm{g})$溶于$100~\mathrm{l}$ PBS缓冲液进行皮下注射。为避免佐剂引入的免疫调节偏差[47, 48], 实验未使用佐剂。分别在免疫第10天和第20天加强注射同等剂量蛋白( $10~\mathrm{g}$ in $100~\mathrm{l}$ PBS)。对照组小鼠在相同部位注射等量PBS。初次免疫30天后处死小鼠分离脾细胞用于其他功能检测。

在另一组独立实验中, BALB/c小鼠(雌性, $^{8-}$12周龄, 20-25g)购自印度诺伊达国家生物研究所, 随机分为三组( $\overset{\cdot}{n}=6/$组)。分别通过腹腔注射PBS、$\mathrm{Ms_Vc}_{}$或Ms_Rv1507A( $\mathrm{1}\times\mathrm{10^{7}}$ CFU)评估该蛋白的抗原性和免疫原性[49-51]。另设气管内感染组模拟气源性感染, 观察肺部大体病理变化(方法如下)。除细菌通过气管内滴注直接递送至肺表面外, 动物随机分组及处理方式与上述腹腔感染实验相同。

Human Subjects

人类受试者

All experiments involving samples from human subjects were approved by Institutional Ethics Committee (IEC), National Institute of Pathology, New Delhi, India. Informed consent was obtained from patient and healthy individuals (controls) included in the study. Blood samples were collected from treatment naïve, fresh pulmonary TB patients $\left(n=31\right)$ and healthy individuals $\mathrm{\Delta}n=19\mathrm{\Delta}$ ) from All India Institute of Medical Sciences (AIIMS), New Delhi, India. Diagnosis of TB was primarily based on microscopic examination of presence of acid-fast bacilli in sputum smear and chest radio graph y of patients. Healthy individuals with unknown TST/IGRA status were included as controls. Considering $40%$ of the Indian population to be latently infected (52), we assume about 8 individuals out of 19 controls to be latently infected. Exclusion criteria in both groups included HIV or HBsAg positivity along with any co-morbid disease. Healthy controls enrolled in study had no known history of any contact with TB patients or samples.

所有涉及人类受试者样本的实验均获得印度新德里国家病理学研究所机构伦理委员会 (IEC) 批准。研究纳入的患者及健康个体（对照组）均签署知情同意书。血液样本采集自印度新德里全印医学科学研究院 (AIIMS) 的初治肺结核患者 $\left(n=31\right)$ 和健康个体 $\mathrm{\Delta}n=19\mathrm{\Delta}$ )。结核病诊断主要依据痰涂片抗酸杆菌镜检结果及患者胸部X光检查。未进行结核菌素皮肤试验 (TST)/γ干扰素释放试验 (IGRA) 检测的健康个体纳入对照组。考虑到印度约 $40%$ 人口存在潜伏感染 [52]，我们假设19名对照组中约有8人为潜伏感染者。两组排除标准包括HIV或HBsAg阳性以及任何合并症。参与研究的健康对照组均无明确结核病患者或样本接触史。

Splenocyte Isolation

脾细胞分离

Mice were sacrificed after 30 days of primary immunization. Spl en omega ly was evident in animals infected with Ms_Rv1507A. For in-vitro assays, spleen cells from BALB/c mice were obtained using standard protocols (45). Briefly, spleen was isolated, crushed gently, and perfused using 26-gauge needle. The cell suspension was then passed through a cell strainer, centrifuged, and re suspended in RBC lysis buffer $(0.84%\mathrm{NH}_{4}\mathrm{Cl}$ solution) for a minute. The final clean preparation of sp leno cyte s, devoid of erythrocytes, were centrifuged and re-suspended in complete media. Sp leno cyte s $(1~\times~10^{6})$ from all the groups were re-stimulated with recombinant protein $(10\mathrm{g/ml})$ and cultured for $^{48\mathrm{h}}$ . The super nat ant was collected and stored for further analysis.

初次免疫30天后处死小鼠。感染Ms_Rv1507A的动物脾脏明显肿大。体外实验采用标准流程获取BALB/c小鼠脾细胞[45]：分离脾脏后轻柔碾碎，用26G针头灌注，细胞悬液经细胞筛过滤、离心，用红细胞裂解液$(0.84%\mathrm{NH}_{4}\mathrm{Cl}$溶液)重悬1分钟。最终获得的去红细胞脾细胞悬液经离心后重悬于完全培养基中。各组脾细胞$(1~\times~10^{6})$用重组蛋白$(10\mathrm{g/ml})$再刺激培养$^{48\mathrm{h}}$，收集上清液保存待测。

Estimation of IgG Level Against Rv1507A in Mice and Human Sera

小鼠和人类血清中抗Rv1507A IgG水平的测定

Estimation of IgG response in mice and reactivity of human sera against Rv1507A was assessed using ELISA (53). Briefly, 96 well-plates were coated with purified recombinant Rv1507A protein $(10\mathrm{g/ml})$ and kept at $\bar{4}^{\circ}\mathrm{C}$ overnight. The plates were thrice washed with PBST (1X PBS pH 7.2, $0.05%$ tween20) and then blocked with $10%$ FBS for $^{1\mathrm{h}}$ at room temperature. Serum samples, obtained from mice immunized with Rv1507A protein and from mice immunized with Ms_Vc or Ms_Rv1507A, were added at 1:100 dilution to each well and incubated for $2\mathrm{h}$ . Goat anti-mouse IgG-HRP secondary antibody (Merck Limited, Mumbai, India) was added at 1:10000 dilution for $^{1\mathrm{h}}$ (54). After five washes, TMB substrate was added, reaction was stopped with 2N $\mathrm{H}{2}\mathrm{SO}{4}$ , and absorbance was measured at $450\mathrm{nm}$ in a spectrophotometer to calculate the amount of serum IgG level. To assess the specificity of antibodies in patient sera against Rv1507A, the protein-coated plates were incubated with patient serum samples at pre-standardized 1:200 dilutions that provides optimal signal: noise ratio. HRP conjugated secondary antibody was added at 1:10000 dilution. SIGMA FAST TM OPD tablets were used as a substrate. The reaction was finally stopped by adding $50~\mathrm{l}$ of 3N $\mathrm{H}{2}\mathrm{SO}_{4}$ . The optical density was measured at a wavelength of $492\mathrm{nm}$ . Diagnostic values of IgG responses was examined by analysis of Receiver-operating characteristic (ROC) curve to determine the test performance in terms of sensitivity and specificity.

通过ELISA法评估了小鼠IgG反应及人血清对Rv1507A的反应性[53]。简言之，96孔板包被纯化重组Rv1507A蛋白$(10\mathrm{g/ml})$，$\bar{4}^{\circ}\mathrm{C}$过夜。PBST(1X PBS pH 7.2, $0.05%$ tween20)洗涤三次后，室温下用$10%$ FBS封闭$^{1\mathrm{h}}$。分别加入经Rv1507A蛋白免疫小鼠、Ms_Vc或Ms_Rv1507A免疫小鼠的血清样本(1:100稀释)，孵育$2\mathrm{h}$。加入1:10000稀释的山羊抗小鼠IgG-HRP二抗(Merck Limited, Mumbai, India)孵育$^{1\mathrm{h}}$[54]。洗涤五次后加入TMB底物，2N $\mathrm{H}{2}\mathrm{SO}{4}$终止反应，分光光度计$450\mathrm{nm}$测吸光度计算血清IgG水平。评估患者血清抗Rv1507A抗体特异性时，采用预标准化1:200稀释比例(最佳信噪比)孵育蛋白包被板，加入1:10000稀释HRP标记二抗，以SIGMA FAST TM OPD片剂为底物，$50~\mathrm{l}$ 3N $\mathrm{H}{2}\mathrm{SO}_{4}$终止反应，$492\mathrm{nm}$测光密度。通过受试者工作特征(ROC)曲线分析IgG反应的诊断价值，确定检测的敏感性和特异性。

Estimation of Cytokine Levels

细胞因子水平估计

RAW264.7 cells were cultured with different concentrations of Rv1507A protein. Additionally, sp leno cyte s from mice infected with $\mathrm{Ms_Vc}$ or Ms_Rv1507A cells/well) were also seeded in 96-well plate, stimulated with different concentration of recombinant protein Rv1507A and incubated at $37^{\circ}\mathrm{C}$ for 12, 24, and $48\mathrm{h}$ . The levels of secreted cytokines TNF-α, IL-6, and IL12, were quantified using murine standard ELISA Development Kit (PeproTech, Rocky Hill, NJ, USA) as per the manufacturer protocol. Sp leno cyte s from mice immunized with recombinant Rv1507A were in-vitro cultured and stimulated and IFNγ was evaluated in culture super nat ants by ELISA. Briefly, 96 well ELISA plates were coated with $100~\mathrm{l}$ of capture antibody and incubated at room temperature overnight. Plates were washed with $300~\mathrm{l}$ PBST and treated with $1%$ BSA, used as blocking buffer. After $1.5\mathrm{h}$ of blocking, the plates were again washed with PBST and $100~\mathrm{l}$ of super nat ant were added for $2\mathrm{h}$ . After 5 washes with PBST, $100~\mathrm{l}$ of detection antibody was added followed by addition of enzyme-avidin HRP conjugate (100 $\mathrm{{l/well})}$ and TMB substrate ( $100\mathrm{l/}$ well) for color development.

RAW264.7细胞与不同浓度的Rv1507A蛋白共培养。同时，将感染$\mathrm{Ms_Vc}$或Ms_Rv1507A的小鼠脾淋巴细胞($0.1\times10^{6}$个细胞/孔)接种于96孔板，用不同浓度的重组蛋白Rv1507A刺激，并于$37^{\circ}\mathrm{C}$培养12、24和$48\mathrm{h}$。采用小鼠标准ELISA试剂盒(PeproTech, USA)检测培养上清中TNF-α、IL-6和IL-12的分泌水平。重组Rv1507A免疫小鼠的脾淋巴细胞经体外培养刺激后，通过ELISA检测培养上清中IFNγ水平。简述步骤如下：96孔ELISA板每孔加入$100~\mathrm{l}$捕获抗体，室温包被过夜；PBST洗涤($300~\mathrm{l}$)后，用$1%$BSA封闭$1.5\mathrm{h}$；再次PBST洗涤，每孔加入$100~\mathrm{l}$上清孵育$2\mathrm{h}$；PBST洗涤5次后，依次加入$100~\mathrm{l}$检测抗体、酶标亲和素HRP偶联物(100$\mathrm{l/孔}$)和TMB底物($100\mathrm{l/孔}$)显色。

The reaction was stopped using 2N $\mathrm{H}{2}\mathrm{SO}_{4}$ and absorbance observed at $450\mathrm{nm}$ and reference wavelength at $570\mathrm{nm}$ .

反应通过加入2N $\mathrm{H}{2}\mathrm{SO}_{4}$ 终止，并在 $450\mathrm{nm}$ 处测定吸光度，参比波长为 $570\mathrm{nm}$。

Measurement of ROS, NO Levels, and Apoptosis in Macrophage

巨噬细胞中ROS、NO水平及凋亡的测定

RAW 264.7 cells $(2\times10^{5})$ were infected with Ms_Rv1507A or $\mathrm{Ms_Vc}$ at MOI 1:10 in incomplete DMEM for $^\mathrm{4h}$ at $37^{\circ}C$ Cells were washed thrice with PBS and incubated in complete media supplemented with gentamycin. After 12, 24, and $48\mathrm{{h},}$ cells were harvested and washed with PBS. These cells were examined for levels of ROS or NO generated and apoptosis, as mentioned below. For assessing levels of reactive oxygen species (ROS) in RAW264.7 cells, Cell ROX orangeTM (Thermo Fischer Scientific India Pvt Ltd, Mumbai, India) was added and cells were incubated for $30\mathrm{min}$ at $37^{\circ}\mathrm{C}$ . Stained cells were acquired in FACS Canto II cytometer (BD Bioscience s) and data analyzed using FlowJoTM software (Becton, Dickinson and Company, New Jersey, US).

RAW 264.7细胞 $(2\times10^{5})$ 以1:10的感染复数(MOI)在不完全DMEM培养基中分别感染Ms_Rv1507A或$\mathrm{Ms_Vc}$，于$37^{\circ}C$培养$^\mathrm{4h}$。用PBS洗涤细胞三次后，置于含庆大霉素的完全培养基中继续培养。分别在12、24和$48\mathrm{{h}}$后收集细胞并用PBS洗涤。如下所述检测这些细胞的活性氧(ROS)、一氧化氮(NO)生成水平及凋亡情况。

为测定RAW264.7细胞中活性氧(ROS)水平，加入Cell ROX orangeTM试剂(Thermo Fischer Scientific India Pvt Ltd, 印度孟买)，于$37^{\circ}\mathrm{C}$孵育$30\mathrm{min}$。染色细胞通过FACS Canto II流式细胞仪(BD Biosciences)获取数据，并使用FlowJoTM软件(美国新泽西州Becton, Dickinson and Company)进行分析。

The level of NO generated by macrophages was assessed using Griess reagent as per manufacturer’s protocol. Briefly, 100 $\left|\mathfrak{u}\right|$ of Griess reagent $[1%$ sul fan il amide in $2.5% \mathrm{H}{3}\mathrm{PO}{4}$ and $0.1%$ nap h tyl ethylene dia mine in $2.5%$ $\mathrm{H}{3}\mathrm{PO}_{4}]$ was added to $100~\mathrm{l}$ of culture super nat ants and absorbance was measured spec tro photometric ally at $570\mathrm{nm}$ . Apoptosis was assessed using AnnexinV-7AAD staining kit (Biolegend, California, US) according to the manufacturer’s instructions. Briefly, cells treated with Ms_Vc or Ms_Rv1507A were harvested, washed with cold PBS, and re suspended in binding buffer. Cells were stained with Annexin-7AAD stain, incubated for $15\mathrm{min}$ , and analyzed through flow cytometer FACS Canto $\mathrm{II}$ cytometer (BD Bioscience s) using FlowJoTM software. Cells treated with St a uro spor in e $(500\mathrm{nM})$ were used as positive control.

巨噬细胞产生的NO水平采用Griess试剂按制造商说明书进行测定。简言之，将100 $\left|\mathfrak{u}\right|$ Griess试剂 [$1%$ 磺胺溶于 $2.5% \mathrm{H}{3}\mathrm{PO}{4}$ 与 $0.1%$ 萘乙二胺溶于 $2.5%$ $\mathrm{H}{3}\mathrm{PO}_{4}$] 加入 $100~\mathrm{l}$ 培养上清液，于 $570\mathrm{nm}$ 波长处测定吸光度。细胞凋亡采用AnnexinV-7AAD染色试剂盒（美国Biolegend公司）按说明书检测：经Ms_Vc或Ms_Rv1507A处理的细胞经冷PBS洗涤后重悬于结合缓冲液，Annexin-7AAD染色孵育 $15\mathrm{min}$ 后，通过FACS Canto $\mathrm{II}$ 流式细胞仪（BD Biosciences）结合FlowJoTM软件分析。以星形孢菌素 $(500\mathrm{nM})$ 处理组作为阳性对照。

Extracellular Staining of Surface Markers

细胞表面标志物的胞外染色

RAW 264.7 cells or sp leno cyte s $(0.5\times10^{6})$ from mice infected with $\mathrm{Ms_Vc}$ or Ms_Rv1507A were seeded in a 12 well plate, re-stimulated in absence or presence of recombinant Rv1507A protein $(10\mathrm{g/ml})$ for $^{48\mathrm{h}}$ . At the end of incubation, cells were washed with FACS buffer $(\mathrm{PBS}+2%$ FBS) and treated with CD16/32. For surface staining, cells were treated with anti-mouse CD3, CD69, F4/80, CD19, CD4, CD8, CD86, CD80, MHCI, and MHCII antibodies and incubated on ice for $20\mathrm{min}$ . Cells were fixed using $4%$ para formaldehyde. For assessing memory response, sp leno cyte s from mice infected with Ms_Vc or Ms_Rv1507A were incubated in absence and presence of Rv1507A protein $(10\mathrm{g/ml})$ for $96\mathrm{h}$ . Cells were washed, harvested, and stained with fluorescent antibodies against CD44 and CD62L memory markers. At least 20,000 live events were acquired through flow cytometer (BD L SR For tessa) and analyzed using FlowJoTM software.

RAW 264.7细胞或小鼠感染$\mathrm{Ms_Vc}$或Ms_Rv1507A的脾细胞$(0.5\times10^{6})$接种于12孔板，在重组Rv1507A蛋白$(10\mathrm{g/ml})$存在或不存在的情况下重新刺激$^{48\mathrm{h}}$。孵育结束后，用FACS缓冲液$(\mathrm{PBS}+2%$ FBS)洗涤细胞，并用CD16/32处理。表面染色时，细胞用抗小鼠CD3、CD69、F4/80、CD19、CD4、CD8、CD86、CD80、MHCI和MHCII抗体处理，冰上孵育$20\mathrm{min}$。细胞用$4%$多聚甲醛固定。为评估记忆反应，Ms_Vc或Ms_Rv1507A感染小鼠的脾细胞在Rv1507A蛋白$(10\mathrm{g/ml})$存在或不存在的情况下孵育$96\mathrm{h}$。洗涤、收集细胞后用抗CD44和CD62L记忆标记的荧光抗体染色。通过流式细胞仪(BD L SR For tessa)获取至少20,000个活细胞事件，并使用FlowJoTM软件分析。

Intracellular Staining for Cytokines

细胞内细胞因子染色

Sp leno cyte s $(1~\times~10^{6})$ were seeded in 12-well plate and restimulated with Rv1507A $(10\mathrm{g/ml})$ for $^{8\mathrm{h}}$ in presence of Golgi plugTM a nd Golgi stopTM (BD Bioscience s, San Diego, CA, USA). Cells were collected, washed with PBS, and stained with antiCD4 and anti-CD8. Cells were fixed with $4%$ para formaldehyde, per me abl i zed in $0.02%$ triton X-100, followed by washing, and thereafter stained with anti-IFN $\gamma$ and anti-TNF $\alpha$ for $^{1\mathrm{h}}$ . Events were acquired by FACS and analyzed, as mentioned above.

将脾细胞 $(1~\times~10^{6})$ 接种于12孔板中，在Golgi plugTM和Golgi stopTM (BD Biosciences, San Diego, CA, USA) 存在下，用Rv1507A $(10\mathrm{g/ml})$ 再刺激 $^{8\mathrm{h}}$。收集细胞后用PBS洗涤，并用抗CD4和抗CD8抗体染色。细胞经 $4%$ 多聚甲醛固定，$0.02%$ Triton X-100透化，洗涤后与抗IFN-$\gamma$ 和抗TNF-$\alpha$ 抗体孵育 $^{1\mathrm{h}}$。如上所述，通过流式细胞仪获取事件并进行分析。

Mycobacteria l Survival Assay

分枝杆菌存活试验

Mycobacterium smegmatis $\mathrm{m}c^{2}155$ grown till log phase was diluted at 1:100 in 7H9 media. Cells were cultured up to $12\mathrm{h}$ until $\mathrm{OD}{600}$ reached 0.05. Re-inoculated cells were then allowed to grow for $30\mathrm{h}$ and the surviving cells were grown on 7H10 media after every $^{3\mathrm{h}}$ in culture. $\mathrm{OD}_{600}$ was also taken after every $^{3\mathrm{h}}$ up to $30\mathrm{{h}}$ .

耻垢分枝杆菌 $\mathrm{m}c^{2}155$ 在对数生长期按1:100比例稀释于7H9培养基中。细胞培养至 $12\mathrm{h}$ 使 $\mathrm{OD}{600}$ 达到0.05后，将重新接种的细胞继续培养 $30\mathrm{h}$，期间每间隔 $^{3\mathrm{h}}$ 取存活细胞在7H10培养基上培养。同时每 $^{3\mathrm{h}}$ 测定一次 $\mathrm{OD}_{600}$ 值，持续监测至 $30\mathrm{{h}}$。

RAW 264.7 cells were infected with Ms_Vc or Ms_Rv1507A at multiplicity of infection (MOI) of 1:10 for $^\mathrm{4h}$ . The infected macrophages were washed 3 times and incubated in complete media supplemented with gentamicin. Cells were lysed in $1\mathrm{ml}$ of $0.025%$ SDS at various time points and aliquots of appropriate dilutions were plated on 7H11 agar plates. After 3 days of incubation, colonies were counted and survival rate was calculated as compared to the control. In a separate experiment, M. smegmatis $\mathrm{m}c^{2}155$ grown till log phase was diluted at 1:100 in 7H9 media. Cells were cultured up to $12\mathrm{h}$ until $\mathrm{OD}{600}$ reached 0.2. Cells were re-inoculated in presence of $\mathrm{H}{2}\mathrm{O}{2}$ (5 and $10\mathrm{mM}$ ) for 3, 6, or $^{9\mathrm{h}}$ , and the surviving cells were grown on 7H10 media. In a separate experiment to test the susceptibility of strains to nitro s at ive stress, Ms_Rv1507A or $\mathrm{Ms_Vc}$ were incubated with 5 or $10\mathrm{mM}$ $\mathrm{NaNO}_{2}$ for $^{3\mathrm{h}}$ , $^{6\mathrm{h}}$ , or $9\mathrm{h}$ and surviving cells were grown on 7H10 media.

RAW 264.7细胞以1:10的感染复数(MOI)分别感染Ms_Vc或Ms_Rv1507A $^\mathrm{4h}$。感染后的巨噬细胞经三次洗涤后，置于含庆大霉素的完全培养基中培养。在不同时间点用$1\mathrm{ml}$ $0.025%$ SDS裂解细胞，取适量稀释液涂布于7H11琼脂平板。培养3天后计数菌落数，并计算相对于对照组的存活率。

在另一实验中，将对数生长期的耻垢分枝杆菌$\mathrm{m}c^{2}155$按1:100比例稀释于7H9培养基，培养至$\mathrm{OD}{600}$达0.2（最长$12\mathrm{h}$）。将细胞重悬于含$\mathrm{H}{2}\mathrm{O}_{2}$（5或$10\mathrm{mM}$）的培养基中培养3、6或$^{9\mathrm{h}}$，存活细胞接种于7H10培养基。

另设实验检测菌株对亚硝化应激的敏感性：将Ms_Rv1507A或$\mathrm{Ms_Vc}$与5或$10\mathrm{mM}$ $\mathrm{NaNO}_{2}$共孵育$^{3\mathrm{h}}$、$^{6\mathrm{h}}$或$9\mathrm{h}$后，存活细胞接种于7H10培养基。

Mycobacteria l Phagocytosis Assay

分枝杆菌吞噬实验

Recombinant M. smegmatis $\begin{array}{r l r}{(100}&{{}\times}&{10^{6})}\end{array}$ $\mathrm{Ms_Vc}$ and $\mathrm{Ms}{-}\mathrm{Rv}1507\mathrm{A}$ were stained with SYTO-9 $(10\mathrm{M})$ (Thermo scientific) for $45\mathrm{min}$ . The stained cells were washed thrice with PBS to remove excess dye. RAW 264.7 cells were seeded in a 12 well-plate at $(0.2\times10^{6})$ ) and co-cultured with SYTO-9 stained $\mathrm{Ms_Vc}$ or Ms_Rv1507A at MOI 1:10 in incomplete DMEM for $^\mathrm{4h}$ at $37^{\circ}C.$ Cells were washed thrice with PBS and the SYTO-9 stained $\mathrm{Ms_Vc}$ or Ms_Rv1507A internalized by RAW 264.7 cells were analyzed through flow cytometer at various time points. For visualization of phagocytosis, RAW 264.7 cells were seeded on coverslips and allowed to adhere at $37^{\circ}\mathrm{C}$ in $\mathrm{CO}{2}$ incubator. SYTO-9 stained cells were co-cultured with Ms_Vc or $\mathrm{Ms}{-}\mathrm{Rv}1507\mathrm{A}$ at MOI 1:10 in incomplete DMEM for $4\mathrm{h}$ at $37^{\circ}C$ The medium was aspirated, cells were fixed with PFA $2%$ in PBS) for $30\mathrm{min}$ . Excess PFA was quenched with 50 mM $\mathrm{NH_{4}C l}$ in PBS and visualized through fluorescence microscope (Nikon Carl Zeiss) (55).

重组耻垢分枝杆菌 $\begin{array}{r l r}{(100}&{{}\times}&{10^{6})}\end{array}$ $\mathrm{Ms_Vc}$ 和 用 SYTO-9 $(10\mathrm{M})$ (Thermo Scientific) 染色 $45\mathrm{min}$。染色后的细胞用 PBS 洗涤三次以去除多余染料。将 RAW 264.7 细胞以 $(0.2\times10^{6})$ 接种于 12 孔板中，并与 SYTO-9 染色的 $\mathrm{Ms_Vc}$ 或 Ms_Rv1507A 以 MOI 1:10 在不完全 DMEM 中共培养 $4\mathrm{h}$，温度 $37^{\circ}C$。用 PBS 洗涤细胞三次，并通过流式细胞仪在不同时间点分析被 RAW 264.7 细胞内化的 SYTO-9 染色 $\mathrm{Ms_Vc}$ 或 Ms_Rv1507A。为观察吞噬作用，将 RAW 264.7 细胞接种于盖玻片上并在 $\mathrm{CO}{2}$ 培养箱中 $37^{\circ}\mathrm{C}$ 贴壁。SYTO-9 染色的细胞与 Ms_Vc 或 $\mathrm{Ms}{-}\mathrm{Rv1507\mathrm{A}}$ 以 MOI 1:10 在不完全 DMEM 中共培养 $4\mathrm{h}$，温度 $37^{\circ}C$。吸弃培养基，用 PBS 配制的 $2%$ PFA 固定细胞 $30\mathrm{min}$。用 50 mM $\mathrm{NH_{4}C l}$ 的 PBS 溶液淬灭多余 PFA，并通过荧光显微镜 (Nikon Carl Zeiss) 观察 (55)。

His to logical Analysis of Lungs

肺部组织学分析

BALB/c mice $(n=6)$ ) were injected with either Phosphate buffer saline (un-infected) or $\mathrm{Ms_Vc}$ $(1\times10^{7}$ CFU) or $\mathrm{Ms}_{-}\mathrm{Rv}1507\mathrm{A}$ cells $(1~\times 10^{7}$ CFU) intra-tracheally. The lungs from representative mice were removed after 30 days of infection and fixed with $4%$ para formaldehyde in phosphate-buffered saline (PBS). For his to pathological examination the lungs were sliced into $1.5~\times~1.0\mathrm{cm}$ slices with a surgical scalpel so that the thickness of the tissue was $5\mathrm{mm}$ . Each specimen was then labeled separately and then transferred to the automated tissue processor (Microm International GmbH, Germany). The tissue processor automatically processed the lung and spleen tissue to 12 cycles overnight which included 1 change in $10%$ formalin followed by graded dehydration in 2 changes each in $70%$ alcohol, $80%$ alcohol, $90%$ alcohol, respectively. The tissue was transferred to absolute alcohol to complete the dehydration. It was then subjected to 2 changes each in liquid chloroform for clearing followed by 2 changes with molten paraffin for embedding. The paraffin embedded tissues were then processed in TEC2800 cry o console for making blocks. The formalin-fixed, paraffin embedded blocks were sectioned into $4\mathrm{m}$ ribbons using rotary microtome (Leica Biosystems Inc., USA). The sections were taken on floatation bath and placed over glass slides for staining and then rehydrated with $90%$ alcohol, $80%$ alcohol, $70%$ alcohol, respectively, and brought into distilled water. The sections were finally stained with he mato xy lin and eosin for evaluation.

BALB/c小鼠 $(n=6)$ ) 分别通过气管内注射磷酸盐缓冲盐水(未感染组)、$\mathrm{Ms_Vc}$ $(1\times10^{7}$ CFU)或细胞 $(1~\times 10^{7}$ CFU)。感染30天后取代表性小鼠肺组织，用磷酸盐缓冲盐水(PBS)配制的$4%$多聚甲醛固定。为进行组织病理学检查，用手术刀将肺组织切成$1.5~\times~1.0\mathrm{cm}$切片，厚度为$5\mathrm{mm}$。每个标本单独标记后转移至全自动组织处理仪(Microm International GmbH, Germany)。该仪器通过12道程序对肺脾组织进行过夜处理：先用$10%$福尔马林处理1次，随后依次用$70%$、$80%$、$90%$乙醇各脱水2次，最后用无水乙醇完成脱水。接着用液态氯仿透明化处理2次，熔融石蜡包埋2次。石蜡包埋组织经TEC2800冷冻台制成组织块后，使用旋转式切片机(Leica Biosystems Inc., USA)将福尔马林固定石蜡块切成$4\mathrm{m}$薄片。切片经浮水浴贴附于载玻片，依次用$90%$、$80%$、$70%$乙醇复水至蒸馏水，最终进行苏木精-伊红染色评估。

Statistical Analysis

统计分析

Statistical analysis was performed using GraphPad Prism8 software. Statistical significance was determined using ANOVA and Mann–Whitney test. $\textit{P}<0.05$ was considered significant, $^{}p<0.05$ , $^{}p<0.01$ , $^{}p<0.001$ , and $^{****}p$ $<~0.0001$ . ROC curve analysis and the area under the curve (AUC) with $95%$ CI for Rv1507A was also calculated using GraphPad Prism. The optimal cut-off value was also determined from the ROC curve at maximal specificity and sensitivity to determine a value that could correctly classify patients and controls.

使用GraphPad Prism8软件进行统计分析。采用ANOVA和Mann-Whitney检验确定统计学显著性，设定$\textit{P}<0.05$为显著水平，并标注$^{}p<0.05$、$^{}p<0.01$、$^{}p<0.001$及$^{****}p$ $<~0.0001$。同时通过GraphPad Prism计算Rv1507A的受试者工作特征曲线(ROC曲线)及曲线下面积(AUC)，并给出95%置信区间(CI)。根据ROC曲线在最大特异性和灵敏度处确定最佳截断值，用于准确区分患者与对照组。

RESULTS

结果

In-silico Analysis and Knock-in of Rv1507A in M. smegmatis Reveals Its Mycobacteria l Specificity and Immunogen ic Potential

耻垢分枝杆菌中Rv1507A的计算机模拟分析与基因敲入揭示其分枝杆菌特异性及免疫原性潜力

The hypothetical M. tb protein, Rv1507A, was examined to assess its role in modulating immune response in the host. The ORF of M. tb genomic sequence for possible coding segments that could translate into putative proteins was studied. A comparison of M. tb Rv1507A putative protein with known protein sequences available in database showed that it is absent in other species of mycobacteria including BCG (data not shown). M. tb Rv1507A also exhibited intrinsically disordered regions, but no protein binding sites, and possessed B cell epitopes (Figure S1) pointing to its antigen i city and immunogen i city. The secretory nature was predicted by Predict Protein software, which was further confirmed by western blot analysis of M. tb H37Rv culture filtrate (Figure S2). M. tb Rv1507A was cloned, expressed in pET28a vector, and the recombinant protein so obtained was purified (Figure S3). M. tb Rv1507A gene was also sub-cloned in pST-Ki expression vector and electro po rated in non-pathogenic M. smegmatis. Positive knock-in constructs of M. smegmatis harboring His-tagged Rv1507A (Ms_Rv1507A) or Vector control pST-Ki (Ms_Vc) were cultured for further use (Figure S3).

为评估假设的结核分枝杆菌蛋白Rv1507A在调控宿主免疫应答中的作用，研究人员对该蛋白进行了分析。通过研究结核分枝杆菌基因组序列的开放阅读框(ORF)，鉴定出可能编码推定蛋白的片段。与数据库中的已知蛋白序列对比显示，Rv1507A在包括卡介苗(BCG)在内的其他分枝杆菌物种中不存在（数据未显示）。该蛋白具有内在无序区域但无蛋白结合位点，同时含有B细胞表位（图S1），表明其具有抗原性和免疫原性。通过Predict Protein软件预测其分泌特性，并经结核分枝杆菌H37Rv培养滤液的western blot分析进一步证实（图S2）。研究人员将Rv1507A克隆至pET28a载体表达，并纯化获得重组蛋白（图S3）。此外，将Rv1507A基因亚克隆至pST-Ki表达载体，通过电穿孔转化至非致病性耻垢分枝杆菌。培养获得携带His标签Rv1507A（Ms_Rv1507A）或空载体对照pST-Ki（Ms_Vc）的阳性敲入耻垢分枝杆菌构建体以供后续使用（图S3）。

FIGURE 1 | Rv1507A induces pro-inflammatory cytokines. RAW264.7 cells were treated with purified Rv1507A protein (2, 5, $10{\mathrm{g/m}})$ . LPS was used as positive control while PBS and heat in activated $(\mathsf{H}\mathsf{I})$ protein served as negative controls. Levels of IL-12 (A), IL-6 (B), and TNF $\cdot\alpha$ (C) were estimated using ELISA, as described in methods. Representative data from three experiments show the concentration of IL-12, IL-6, and TNF as mean $\pm$ SEM. Statistical significance was determined (Continued)

图 1 | Rv1507A诱导促炎细胞因子。RAW264.7细胞经纯化Rv1507A蛋白(2, 5, $10{\mathrm{g/m}})$处理。LPS作为阳性对照，PBS和热灭活$(\mathsf{H}\mathsf{I})$蛋白作为阴性对照。采用ELISA法检测IL-12(A)、IL-6(B)和TNF$\cdot\alpha$(C)水平，方法如所述。三项实验的代表性数据显示IL-12、IL-6和TNF浓度为均值$\pm$SEM。统计学显著性判定(待续)

FIGURE 1 | with one-way ANOVA. Additionally, spleen was recovered from BALB/c mice $(n=7)$ that were injected with Ms_Vc $(1\times10^{7})$ or Ms_Rv1507A $(1\times10^{7})$ . Primed sp leno cyte s were cultured $j n$ -vitro in presence of Rv1507A protein. Culture super nat ants were harvested after 12 h, 24 h, and 48 h and concentrations of IL-12 (D), IL-6 (E), and TNF $\cdot\alpha$ (F) were determined. Representative data from triplicate wells show the concentration of IL-12, IL-6, and TNF $\cdot\alpha$ as mean $\pm$ SEM. Statistical significance was determined with two-way ANOVA. $P<0.05$ was considered significant, $^{\star}p<0.05$ , $^{\star\star}p<0.01$ , $^{\star\star\star}p<0.001$ , and $^{\star\star\star}p<0.0001$ (nd-not detected).

图 1 | 采用单因素方差分析。此外，从注射了Ms_Vc $(1\times10^{7})$ 或Ms_Rv1507A $(1\times10^{7})$ 的BALB/c小鼠 $(n=7)$ 中取出脾脏。预激的脾细胞在Rv1507A蛋白存在下进行体外培养 $j n$ 。分别在12小时、24小时和48小时后收集培养上清，测定IL-12 (D)、IL-6 (E)和TNF $\cdot\alpha$ (F)的浓度。三复孔的代表性数据显示IL-12、IL-6和TNF $\cdot\alpha$ 的浓度为均值 $\pm$ 标准误。统计学显著性采用双因素方差分析确定。$P<0.05$ 视为显著，$^{\star}p<0.05$，$^{\star\star}p<0.01$，$^{\star\star\star}p<0.001$，以及 $^{\star\star\star}p<0.0001$ (nd-未检测到)。

M. tb Rv1507A Activates Macrophages and Induces Pro-inflammatory Cytokine Response

M. tb Rv1507A 激活巨噬细胞并诱导促炎细胞因子反应

The role of $M$ . tb putative protein Rv1507A in eliciting cytokine secretion was examined in-vitro by treating RAW264.7 macrophage cells with Rv1507A protein for $^{48\mathrm{h}}$ . The Rv1507A protein, being recombinant ly purified from E.coli BL21(DE3), could possibly contain LPS that may induce cytokine response and generate false results. In order to rule out this possibility, heat in activated (HI) recombinant protein (Rv1507A) was used as control. The super nat ant collected from RAW264.7 cells was assessed for levels of cytokines using ELISA. Rv1507A induced significant production of cytokines IL-12 (Figure 1A), IL-6 (Figure 1B), and TNF (Figure 1C) in macrophages. Moreover, RAW264.7 macrophage cells infected with Ms_Rv1507A also showed increased expression of CD86 (associated with maturation of APCs), CD40, and MHC I (associated with antigen presentation in APCs) (Figure S4). These results point to the ability of Rv1507A to enhance antigen presentation and co-stimulation for evoking an efficient immune response.

$M$ 的作用。通过用 Rv1507A 蛋白处理 RAW264.7 巨噬细胞体外检测了结核分枝杆菌假定蛋白 Rv1507A 在诱导细胞因子分泌中的作用。Rv1507A 蛋白是从大肠杆菌 BL21(DE3) 中重组纯化的，可能含有可诱导细胞因子反应并产生假结果的 LPS。为了排除这种可能性，使用热灭活 (HI) 的重组蛋白 (Rv1507A) 作为对照。使用 ELISA 评估从 RAW264.7 细胞收集的上清液中细胞因子的水平。Rv1507A 在巨噬细胞中显著诱导了细胞因子 IL-12 (图 1A)、IL-6 (图 1B) 和 TNF (图 1C) 的产生。此外，感染 Ms_Rv1507A 的 RAW264.7 巨噬细胞也显示出 CD86 (与 APC 成熟相关)、CD40 和 MHC I (与 APC 中的抗原呈递相关) 的表达增加 (图 S4)。这些结果表明 Rv1507A 能够增强抗原呈递和共刺激，从而引发有效的免疫反应。

In-vivo Exposure to M. tb Rv1507A Also Induces Pro-inflammatory Response and Up-Regulates Molecules Associated With Cell Maturation, Co-stimulation, and Antigen Presentation

体内暴露于M. tb Rv1507A同样会诱导促炎反应并上调与细胞成熟、共刺激及抗原呈递相关的分子

The immuno modulator y role of Rv1507A was assessed by immunizing $\mathrm{BALB/c}$ mice with purified Rv1507A proteins and also by infecting with recombinant $M.$ smegmatis (Ms_Rv1507A). Administration of Ms_Rv1507A resulted in spl en omega ly (Figure S5A) in mice and increase in number of sp leno cyte s (Figure S5B) as compared to $\mathrm{Ms_Vc}$ . Primed sp leno cyte s, obtained from mice infected with Ms_Rv1507A or $\mathrm{Ms_Vc},$ were re-stimulated with Rv1507A proteins $(10\mathrm{g/ml})$ . Culture super nat ant from re-stimulated sp leno cyte s was obtained after $12\mathrm{h}$ , $24\mathrm{{h}}$ , and $48\mathrm{h}$ . Re-stimulation of primed sp leno cyte s with Rv1507A induces significantly higher IL-12 (Figure 1D), IL6 (Figure 1E), and TNF (Figure 1F). These results point to the pro-inflammatory response elicited by Rv1507A protein. The effect of Ms_Rv1507A on generation of lymphocyte sub-populations was evaluated using multi-parameter flow cytometry. Results in Figures 2A,B show that Ms_Rv1507A induces significant $(p<0.01)$ increase in generation of $\mathrm{CD}3^{+}$ T cells, $\mathrm{CD}19^{+}$ B cells, and F4/80 macrophage cells. In order to ascertain the mechanistic basis of immuno modulation by $\mathrm{Ms}_{-}\mathrm{Rv}1507\mathrm{A}$ , early activation marker (CD69), co-sti mula tory receptors (CD80, CD86, and CD40), and antigen presenting molecules (MHC II) were examined. Results in Figure 2C show the mean fluorescence intensity (MFI), as an estimate of surface expression of these molecules on sp leno cyte s. There is significant $(p<0.01)$ increase (Figures 2A,C) in expression of CD80, CD86, MHC II, CD40, and CD69 on specific cell populations in sp leno cyte s from animals infected with Ms_Rv1507A as compared to Ms_Vc. It is evident that M. tb Rv1507A possibly modulates T cell activity through increase in expression of CD80 and CD86. Rv1507A can also modulate the activity of B cells through up regulation of CD40 which acts as co-sti mula tory molecule and is required for association of APCs with T cells during antigen presentation. An increase in MHC II in presence of Ms_Rv1507A points to their possible role in modulating antigen presentation to $\mathrm{CD4^{+}}$ cells.

通过用纯化的Rv1507A蛋白免疫BALB/c小鼠以及感染重组耻垢分枝杆菌(Ms_Rv1507A)来评估Rv1507A的免疫调节作用。与Ms_Vc相比，Ms_Rv1507A感染导致小鼠脾肿大(图S5A)和脾细胞数量增加(图S5B)。用Rv1507A蛋白(10μg/ml)对来自Ms_Rv1507A或Ms_Vc感染小鼠的致敏脾细胞进行再刺激。再刺激后12h、24h和48h收集培养上清。Rv1507A再刺激致敏脾细胞可显著诱导更高水平的IL-12(图1D)、IL-6(图1E)和TNF-α(图1F)，表明Rv1507A蛋白能引发促炎反应。

采用多参数流式细胞术评估Ms_Rv1507A对淋巴细胞亚群生成的影响。图2A,B结果显示Ms_Rv1507A能显著(p<0.01)增加CD3+T细胞、CD19+B细胞和F4/80巨噬细胞的生成。为阐明Ms_Rv1507A免疫调节的机制基础，检测了早期活化标志物(CD69)、共刺激受体(CD80、CD86和CD40)及抗原呈递分子(MHC II)。图2C显示这些分子在脾细胞表面表达的平均荧光强度(MFI)。与Ms_Vc相比，Ms_Rv1507A感染动物脾细胞特定群体中CD80、CD86、MHC II、CD40和CD69的表达均显著(p<0.01)升高(图2A,C)。显然，结核分枝杆菌Rv1507A可能通过增加CD80和CD86表达来调节T细胞活性。Rv1507A还能通过上调共刺激分子CD40来调节B细胞活性，该分子在抗原呈递过程中是APC与T细胞结合所必需的。Ms_Rv1507A存在时MHC II的增加表明其可能在调节向CD4+细胞的抗原呈递中发挥作用。

Rv1507A Exposure Generates Poly functional T Cells and Cells Having Memory Like Phenotype

Rv1507A 暴露可产生多功能 T 细胞及具有记忆样表型的细胞

The above results presented so far pointed to the modulation of T cell responses by Rv1507A. The role of Rv1507A in generating poly functional T cells was studied by infecting mice with Ms_Rv1507A or Ms_Vc or else by immunizing with recombinant purified Rv1507A protein. The primed sp leno cyte s were obtained after 4 weeks and re-stimulated with Rv1507A protein. In response to in-vitro protein stimulation, a significant $(p<0.001)$ increase in number of double-positive (IFN $\cdot\gamma^{+}/\mathrm{TNF}{-\alpha^{+}}$ ) $\mathrm{CD4^{+}}$ cells (Figures 3A,B) as well as $\mathrm{CD}8^{+}$ cells $(p<0.05)$ (Figures 3C,D) was observed in immunized mice. These observations demonstrate that Rv1507A induces antigen-specific multi functional $\mathrm{CD4^{+}/C D8^{+}}$ T cells in mice, thereby pointing to its role in modulating T cells function in response to infections. This observation was validated by estimation of $\operatorname{IFN-}\gamma$ from culture super nat ants, which revealed enhanced secretion of this classical Th1 cytokine from Rv1507A immunized animals (Figure S6). Sub-populations of $\mathrm{CD4^{+}}$ or $\mathrm{CD}8^{+}$ cells expressing CD44 and CD62L were examined by flow cytometry. Rv1507A induced significant $(p$ $<0.05$ memory like response with increase in central memory response $\mathrm{^{\prime}C D8^{+}C D44^{+}C D62L^{+}}$ T cells, $\mathrm{CD4^{+}C D44^{+}C D62L^{+}}$ T cells) and effector memory $(\mathrm{CD4^{+}C D44^{+}}$ T cells, $\mathrm{CD8^{+}C D44^{+}}$ T cells) as shown in Figures 4A,B, respectively. Consistent with the above results, $\mathrm{Ms}_{-}\mathrm{Rv}1507\mathrm{A}$ also induced strong central (Figures 4C,E) and effector memory response (Figures 4D,F).

上述结果表明Rv1507A对T细胞应答具有调节作用。通过用Ms_Rv1507A或Ms_Vc感染小鼠，或使用重组纯化Rv1507A蛋白免疫小鼠，研究了Rv1507A在产生多功能T细胞中的作用。4周后获取致敏脾细胞，并用Rv1507A蛋白再刺激。体外蛋白刺激结果显示：免疫小鼠中双阳性(IFN-γ⁺/TNF-α⁺) CD4⁺细胞(图3A,B)和CD8⁺细胞(图3C,D)数量显著增加(p<0.001和p<0.05)，表明Rv1507A能诱导小鼠产生抗原特异性多功能CD4⁺/CD8⁺ T细胞，提示其在调节感染应答中T细胞功能的作用。这一结果通过培养上清液中IFN-γ的检测得到验证，Rv1507A免疫组动物该经典Th1细胞因子分泌增强(图S6)。流式细胞术分析显示，Rv1507A可显著(p<0.05)诱导记忆样应答，增加中枢记忆应答(CD8⁺CD44⁺CD62L⁺ T细胞、CD4⁺CD44⁺CD62L⁺ T细胞)和效应记忆(CD4⁺CD44⁺ T细胞、CD8⁺CD44⁺ T细胞)，分别如图4A,B所示。与上述结果一致，Ms_Rv1507A也诱导了强劲的中枢记忆(图4C,E)和效应记忆应答(图4D,F)。

M. tb Rv1507A Induces IgG Response That Exhibits Sero-Specificity

M. tb Rv1507A 诱导具有血清特异性的 IgG 反应

Having shown the ability of Rv1507A to generate poly functional T cells as well as memory cells, we further assessed its effect on generation of humoral immune response. The immunoglobulin

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在证实Rv1507A能诱导多功能T细胞和记忆细胞生成后，我们进一步评估了其对体液免疫应答的影响。免疫球蛋白

FIGURE 2 | Rv1507A induces enhanced generation of lymphoid sub populations. BALB/c mice $(n=6)$ were injected with Ms_Vc $(1\times10^{7})$ or Ms_Rv1507A $(1\times10^{7})$ . Primed sp leno cyte s obtained from mice were cultured $j n$ -vitro in presence of Rv1507A. The generation of $\mathrm{CD}3^{+}$ T cells, $\mathsf{C D}\mathsf{1}\mathsf{9}^{+}$ B cells, and $\mathsf{F4}/80$ macrophage cells, were measured by flow cytometry (A,B). Note the increased generation of $\mathsf{C D}\mathsf{1}\mathsf{9^{+}}$ B cells, $\mathrm{CD}3^{+}$ T cells, and $\mathsf{F4/80^{+}}$ macrophage cells. Expression of CD80, CD86, MHCII, CD40, and CD69 on primed sp leno cyte s (A,C) are shown in MFI as mean $\pm$ SEM. $p<0.05$ was considered significant, $^{\star\star}p<0.01$ .

图 2 | Rv1507A促进淋巴亚群生成增强。BALB/c小鼠$(n=6)$分别注射Ms_Vc$(1\times10^{7})$或Ms_Rv1507A$(1\times10^{7})$。从小鼠获取的致敏脾细胞在Rv1507A存在下进行体外培养($j n$)。通过流式细胞术检测$\mathrm{CD}3^{+}$T细胞、$\mathsf{C D}\mathsf{1}\mathsf{9}^{+}$B细胞和$\mathsf{F4}/80$巨噬细胞的生成情况(A,B)。观察到$\mathsf{C D}\mathsf{1}\mathsf{9^{+}}$B细胞、$\mathrm{CD}3^{+}$T细胞和$\mathsf{F4/80^{+}}$巨噬细胞生成增加。致敏脾细胞表面CD80、CD86、MHCII、CD40和CD69的表达水平(A,C)以MFI(平均荧光强度)表示，数据为均值$\pm$标准误。$p<0.05$视为具有统计学差异，$^{\star\star}p<0.01$。

FIGURE 3 | Rv1507A induces generation of poly-functional $14-\gamma$ and TNF $\cdot\alpha$ secreting $\mathtt{C D4^{+}}$ and $\mathrm{CD8^{+}}$ cells. BALB/c mice were immunized with either purified Rv1507A proteins $(10\mathrm{g/m}|)$ or PBS alone. Sp leno cyte s obtained from mice were cultured in absence or presence of Rv1507A protein $(10\mathrm{g/m}|)$ for 48 h and the intracellular levels of $14\times1-\frac{1}{2}$ and TNF were estimated, as described in methods. The percentage of $\mathtt{C D4^{+}}$ TNF $\alpha^{+}|\mathsf{F}|\mathsf{N}{-}\gamma^{+}$ cells (A) and $\mathtt{C D8^{+}}$ TNF $\mathsf{a}^{+}\mathsf{I F N{-}}\gamma^{+}$ cells (C) are shown as mean $\pm$ SEM. Representative corresponding density plots of $\mathtt{C D4^{+}}$ TNF $\alpha^{+}$ IFN $\gamma^{+}$ cells (B) and $\mathtt{C D8^{+}}$ TNF $\alpha^{+}|\mathsf{F N-\gamma}\gamma^{+}$ cells (D) are shown. Statistical significance was determined with one tailed Mann–Whitney test. $p<0.05$ was considered significant, $^{\star}p<0.05$ , $^{\star\star\star}p<0.001$ .

图 3 | Rv1507A诱导产生分泌$14-\gamma$和TNF$\cdot\alpha$的多功能$\mathtt{C D4^{+}}$和$\mathrm{CD8^{+}}$细胞。BALB/c小鼠分别接种纯化Rv1507A蛋白$(10\mathrm{g/m}|)$或单独PBS。从小鼠获取的脾细胞在无或有Rv1507A蛋白$(10\mathrm{g/m}|)$条件下培养48小时，细胞内$14\times1-\frac{1}{2}$和TNF水平按方法所述进行检测。$\mathtt{C D4^{+}}$ TNF$\alpha^{+}|\mathsf{F}|\mathsf{N}{-}\gamma^{+}$细胞(A)和$\mathtt{C D8^{+}}$ TNF$\mathsf{a}^{+}\mathsf{I F N{-}}\gamma^{+}$细胞(C)的百分比以均值$\pm$标准误表示。代表性密度图显示$\mathtt{C D4^{+}}$ TNF$\alpha^{+}$ IFN$\gamma^{+}$细胞(B)和$\mathtt{C D8^{+}}$ TNF$\alpha^{+}|\mathsf{F N-\gamma}\gamma^{+}$细胞(D)。统计学显著性采用单尾Mann-Whitney检验判定，$p<0.05$视为显著，$^{\star}p<0.05$，$^{\star\star\star}p<0.001$。

G $\left(\mathrm{IgG}\right)$ response from the sera of mice immunized with purified Rv1507A protein or Rv1507A knock-in M. smegmatis was done using ELISA. Mice treated with Rv1507A protein elicited nearly two-fold higher titer of IgG as compared to control mice. Similarly, mice injected with Ms_Rv1507A elicited nearly two-fold higher IgG response as compared to mice injected with Ms_Vc (Figure 5A). These results point to the immunogen e city of Rv1507A in eliciting IgG response. Following this observation, humoral immune responses directed against the Rv1507A protein were also compared in pulmonary tuberculosis (PTB) patients and healthy controls. Sera of all the PTB patients showed significantly $(p<0.001)$ ) higher IgG reactivity against Rv1507A protein antigen as compared to sera of the healthy controls (Figure 5B). Based on these OD values, an ROC analysis was performed that revealed optimal cut-off for the assay to distinguish patients and controls. At cut-off OD values of 0.947, sensitivity of $96.7%$ with $100%$ specificity was achieved. Area under Curve (AUC) value being 0.996 ( $95%$ CI, 0.988–1.000) revealed an excellent diagnostic efficacy of Rv1507A (Figure 5C). These results point to the specificity of Rv1507A, and also its value as a novel diagnostic marker.

采用ELISA法检测了纯化Rv1507A蛋白或Rv1507A敲入耻垢分枝杆菌免疫小鼠血清中的G$\left(\mathrm{IgG}\right)$应答。与对照组相比，Rv1507A蛋白处理小鼠产生的IgG效价高出近两倍。同样，注射Ms_Rv1507A的小鼠比注射Ms_Vc的小鼠IgG应答水平高出近两倍(图5A)。这些结果表明Rv1507A具有激发IgG应答的免疫原性。基于此发现，我们进一步比较了肺结核(PTB)患者与健康对照者针对Rv1507A蛋白的体液免疫应答。所有PTB患者血清对Rv1507A蛋白抗原的IgG反应性均显著高于健康对照组$(p<0.001)$)(图5B)。根据这些OD值进行ROC分析，确定了区分患者与对照组的最佳临界值。当OD临界值为0.947时，检测灵敏度达$96.7%$，特异性为$100%$。曲线下面积(AUC)值为0.996($95%$CI，0.988-1.000)，表明Rv1507A具有极佳的诊断效能(图5C)。这些结果证实了Rv1507A的特异性，并提示其作为新型诊断标志物的价值。

FIGURE 4 | Rv1507A protein and recombinant Ms_Rv1507A mount effector and central memory like response. Spleens were recovered from BALB/c mice that were either injected with purified Rv1507A proteins $10{\mathrm{g/m}}|)$ or PBS alone. Sp leno cyte s were cultured in presence of Rv1507A, in-vitro for 96 h. Representative (Continued)

图 4 | Rv1507A蛋白和重组Ms_Rv1507A能激发效应记忆和中枢记忆样免疫应答。从注射纯化Rv1507A蛋白(10μg/ml)或单独PBS的BALB/c小鼠中取出脾脏。脾细胞在体外用Rv1507A培养96小时。(续)

FIGURE 4 | density plots of $\mathtt{C D8^{+}C D44^{h i g h}C D62L^{p i g h}}$ $(\top_{\mathsf{C M}})$ cells and $\mathtt{C D4^{+}C D44^{n i g h}C D62L^{p i g h}}$ cells are shown (A). $\mathsf{C D8^{+}C D44^{h i g h}}$ $(\mathsf{T}{\mathsf{E M}})$ cells and $\mathsf{C D4^{+}C D44^{h i g h}}$ cells are shown in (B). Additionally, sp leno cyte s from mice infected with Ms_Vc $(1\times10^{7})$ or Ms_Rv1507A $(1\times10^{7})$ ) were re-stimulated with Rv1507A in-vitro for 96 h. Percent of $\mathtt{C D8^{+}}$ cells expressing CD 44 high CD 62 L high (C) and $\mathtt{C D44^{h i g h}}$ cells (D) were measured by flow cytometry, shown as mean $\pm$ SEM. Similarly $\mathtt{C D4^{+}}$ ${\sf T}{\sf C M}$ and $\mathsf{T}_{\mathsf{E M}}$ were quantified and depicted in (E,F), respectively. Statistical significance was determined with one tailed Mann–Whitney test. $p<0.05$ was considered significant, $^{\star}p<0.05$ , $^{\star\star}p<0.01$ .

图 4 | $\mathtt{C D8^{+}C D44^{h i g h}C D62L^{p i g h}}$ ($\top_{\mathsf{C M}}$) 细胞与 $\mathtt{C D4^{+}C D44^{n i g h}C D62L^{p i g h}}$ 细胞的密度图如 (A) 所示。$\mathsf{C D8^{+}C D44^{h i g h}}$ ($\mathsf{T}{\mathsf{E M}}$) 细胞与 $\mathsf{C D4^{+}C D44^{h i g h}}$ 细胞展示于 (B)。此外，用 Rv1507A 体外重刺激 Ms_Vc $(1\times10^{7})$ 或 Ms_Rv1507A $(1\times10^{7})$ 感染小鼠的脾细胞 96 小时。通过流式细胞术检测表达 CD44 high CD62L high 的 $\mathtt{C D8^{+}}$ 细胞百分比 (C) 和 $\mathtt{C D44^{h i g h}}$ 细胞百分比 (D)，数据以均值 $\pm$ 标准误表示。类似地，$\mathtt{C D4^{+}}$ ${\sf T}{\sf C M}$ 与 $\mathsf{T}_{\mathsf{E M}}$ 分别在 (E,F) 中进行定量展示。统计学显著性采用单尾 Mann-Whitney 检验判定，$p<0.05$ 视为显著，$^{\star}p<0.05$，$^{\star\star}p<0.01$。

Rv1507A Knock-in M. smegmatis Shows Increased Uptake in Macrophages and Associated Stress Responses

Rv1507A敲入耻垢分枝杆菌在巨噬细胞中摄取增加及相关应激反应

Sustained expression of Rv1507A in recombinant M. smegmatis, confirmed by western blot, revealed successful integration (Figure S7A). A comparison of growth kinetics of Ms_Rv1507A and Ms_Vc culture showed no significant difference in doubling time (Figure S7B). Fluorescence microscopy of SYTO-9 stained bacteria showed that increased number of Ms_Rv1507A were localized in the cytoplasm of macrophage cells as compared to Ms_Vc (Figure 6A). The infect iv it y of recombinant Ms_Rv1507A was assessed by their ability to infect cells in a given time as determined by CFU. Higher CFU load suggested that nonpathogenic M. smegmatis acquired infect iv it y by virtue of incorporation and expression of Rv1507A in recombinant $M.$ smegmatis (Figure 6B).

Western blot 证实重组耻垢分枝杆菌中 Rv1507A 的持续表达，显示成功整合（图 S7A）。Ms_Rv1507A 与 Ms_Vc 培养物的生长动力学比较显示倍增时间无显著差异（图 S7B）。SYTO-9 染色细菌的荧光显微镜观察表明，与 Ms_Vc 相比，更多 Ms_Rv1507A 定位于巨噬细胞胞质内（图 6A）。通过 CFU 测定重组 Ms_Rv1507A 在特定时间内感染细胞的能力，评估其感染性。更高的 CFU 载量表明非致病性耻垢分枝杆菌通过重组 $M.$ smegmatis 中 Rv1507A 的整合与表达获得了感染性（图 6B）。

The physiological responses in RAW264.7 cells due to infection with Ms_Rv1507A or Ms_Vc were assessed every $12\mathrm{h}$ by estimating ROS or NO generated and induction of apoptosis. Results in Figure 6C showed that RAW264.7 cells infected with $\mathrm{Ms}_{-}\mathrm{Rv}1507\mathrm{A}$ generated nearly two-fold higher $\begin{array}{r}{\dot{\boldsymbol{P}}<0.001,}\end{array}$ ) ROS at 24 and $48\mathrm{h}$ as compared to infection with Ms_Vc. Results in Figure 6D show that Ms_Rv1507A induces generation of NO, nearly two-fold higher $(p<0.001)$ at $24\mathrm{h}$ and three-fold higher $\mathit{\Omega}^{\prime}p<0.0001)$ at $48\mathrm{{h},}$ as compared to cells infected with Ms_Vc. These results show that upon infection with Rv1507A knock-in M. smegmatis, RAW264.7 cells exhibit stress response through generation of ROS and NO. RAW264.7 cells infected with Ms_Rv1507A also showed reduced viability as compared to cells infected with $\mathrm{Ms_Vc}$ . In order to assess the mechanistic basis of reduction in viability of RAW264.7 cells due to infection with Ms_Rv1507A, cells were stained with AnnexinV-7AAD and percent apoptotic cells were assessed by flow cytometry. Results in Figure 6E show that Ms_Rv1507A induced significant $(p<0.05)$ increase in apoptosis of RAW264.7 cells at $48\mathrm{h}$ post infection as compared to Ms_Vc. The percent apoptotic RAW264.7 cells were not significant at 12 or $24\mathrm{h}$ (data not shown). It is evident that infection with Ms_Rv1507A resulted in stress response in RAW264.7 cells, leading to apoptosis.

通过检测活性氧(ROS)或一氧化氮(NO)的生成及细胞凋亡诱导情况，每$12\mathrm{h}$评估RAW264.7细胞感染Ms_Rv1507A或Ms_Vc后的生理反应。图6C结果显示，与Ms_Vc感染相比，$\mathrm{Ms}_{-}\mathrm{Rv}1507\mathrm{A}$感染的RAW264.7细胞在24和$48\mathrm{h}$时产生近两倍更高的ROS$\begin{array}{r}{\dot{\boldsymbol{P}}<0.001,}\end{array}$。图6D显示Ms_Rv1507A诱导的NO生成量在$24\mathrm{h}$时较Ms_Vc感染组高近两倍$(p<0.001)$，在$48\mathrm{{h},}$时高三倍$\mathit{\Omega}^{\prime}p<0.0001)$。这些结果表明，感染Rv1507A敲入型耻垢分枝杆菌后，RAW264.7细胞通过产生ROS和NO表现出应激反应。与$\mathrm{Ms_Vc}$感染相比，Ms_Rv1507A感染的RAW264.7细胞存活率也降低。为探究Ms_Rv1507A感染导致RAW264.7细胞存活率降低的机制，采用AnnexinV-7AAD染色并通过流式细胞术检测凋亡细胞百分比。图6E显示，与Ms_Vc相比，Ms_Rv1507A在感染后$48\mathrm{h}$显著$(p<0.05)$增加RAW264.7细胞凋亡率，而在12或$24\mathrm{h}$时凋亡率无显著差异（数据未显示）。显然，Ms_Rv1507A感染导致RAW264.7细胞产生应激反应并引发凋亡。

Increased Sur viv ability of Rv1507A Knock-in M. smegmatis Within Macrophage and Other Stress Conditions

Rv1507A敲入耻垢分枝杆菌在巨噬细胞内及其他应激条件下的存活能力增强

The sur viv ability of Ms_Rv1507A within macrophages at different time points, post-infection, was examined by enumerating CFU. The CFU/ml of $\mathrm{Ms}_{-}\mathrm{Rv}1507\mathrm{A}$ or Ms_Vc corresponds to the number of bacteria that survived within the macrophage before being plated on agar plates. Ms_1507A exhibits significantly $(p<0.01)$ ) higher sur viv ability within macrophages for longer durations (Figure 7A). The sur viv ability of Ms_Vc decreased at 24 and $48\mathrm{h}$ of infection compared to Ms_Rv1507A. These results demonstrate that Ms_Rv1507A possess increased sur viv ability within the macrophage. Similarly, RAW264.7 cells infected with Ms_Rv1507A showed about two-fold higher MFI that corresponds to increased uptake of SYTO-9 stained bacteria as compared to $\mathrm{Ms_Vc}$ (Figure S8).

通过计数CFU检测了Ms_Rv1507A在不同感染时间点于巨噬细胞内的存活能力。$\mathrm{Ms}_{-}\mathrm{Rv}1507\mathrm{A}$或Ms_Vc的CFU/ml对应铺板前巨噬细胞内存活细菌数量。Ms_1507A在巨噬细胞内表现出显著$(p<0.01)$的长期存活优势(图7A)。与Ms_Rv1507A相比，Ms_Vc在感染24和$48\mathrm{h}$后存活率下降。这些结果表明Ms_Rv1507A在巨噬细胞内具有更强的存活能力。类似地，感染Ms_Rv1507A的RAW264.7细胞MFI值约为$\mathrm{Ms_Vc}$的两倍，表明SYTO-9染色细菌的摄取量增加(图S8)。

Enhanced levels of ROS and NO in macrophages can effectively get rid of $M.$ . smegmatis infection (56). However, our results show that $\mathrm{Ms}{-}\mathrm{Rv}1507\mathrm{A}$ survives for prolonged duration within the macrophages (Figure 7A). Ms_Vc or Ms_Rv1507A were treated with sodium nitrite (5 and $10\mathrm{mM}$ and hydrogen peroxide (5 and $10\mathrm{mM}$ for up to $^{9\mathrm{h}}$ to induce NO and ROS stress, respectively. The CFU of $\mathrm{Ms_Vc}$ or Ms_Rv1507A as a measure of viable bacteria that survived in the presence of sodium nitrite and hydrogen peroxide were examined. A significantly higher CFU of Ms_Rv1507A as compared to Ms_Vc was observed after treatment of respective cultures with $5\mathrm{mM}$ sodium nitrite for 3 and $9\mathrm{h}$ or with $10\mathrm{mM}$ sodium nitrite for $3,6,$ and $9\mathrm{h}$ (Figure 7B). Similarly, cultures treated with hydrogen peroxide $(5,10\mathrm{mM})$ for 3 and $^{9\mathrm{h}}$ showed significantly higher CFU of $\mathrm{Ms}_{-}\mathrm{Rv}1507\mathrm{A}$ cells as compared Ms_Vc cells (Figure 7C). These results showed that Ms_Rv1507A is more resilient to NO or ROS stress as compared to $\mathrm{Ms_Vc}$ . These results provide a logical interpretation of our earlier results that showed that Ms_Rv1507A survived oxidative stress within macrophages. Therefore, Rv1507A protein aids in survival of mycobacteria under stress conditions generated within immune cells in response to infections.

巨噬细胞中活性氧(ROS)和一氧化氮(NO)水平升高可有效清除耻垢分枝杆菌感染[56]。然而我们的研究结果表明，Ms_Rv1507A能在巨噬细胞内长期存活(图7A)。我们分别用亚硝酸钠(5mM和10mM)和过氧化氢(5mM和10mM)处理Ms_Vc或Ms_Rv1507A长达9小时以诱导NO和ROS应激。通过检测菌落形成单位(CFU)来评估Ms_Vc或Ms_Rv1507A在亚硝酸钠和过氧化氢存在下的存活情况。结果显示，经5mM亚硝酸钠处理3小时和9小时，或10mM亚硝酸钠处理3、6、9小时后，Ms_Rv1507A的CFU均显著高于Ms_Vc(图7B)。类似地，经5mM和10mM过氧化氢处理3小时和9小时的培养物中，Ms_Rv1507A细胞的CFU也显著高于Ms_Vc细胞(图7C)。这些结果表明，与Ms_Vc相比，Ms_Rv1507A对NO或ROS应激具有更强的抵抗力。这为我们先前观察到的Ms_Rv1507A能在巨噬细胞内氧化应激下存活的结果提供了合理解释。因此，Rv1507A蛋白能帮助分枝杆菌在免疫细胞应对感染产生的应激条件下存活。

Intra-tracheal Infection of Ms_Rv1507A Reveals Increased Infiltration of Lymphocytes in the Lungs

气管内感染Ms_Rv1507A显示肺部淋巴细胞浸润增加

After exploring the immunological response of Rv1507A and the increased persistence of Ms_Rv1507A in macrophages along with stress response related correlates, we assessed its effect via aerogenic route to mimic actual infection. His to logical analysis of lung tissue after aerogenic infection revealed enhanced infiltration of lymphocytes into lung parenchyma (Figure 8) in animals infected with Ms_Rv1507A as compared to vector control $(\mathrm{Ms}_\mathrm{Vc})$ . There was no evidence of any granuloma like structure or any caseous necrotic lesions after intra-tracheal in still ation. These observations suggest that Ms_Rv1507A was more immunogen ic as compared to Ms_Vc but was not virulent enough to cause any adverse pathology. All these observations point to the possibility of Rv1507A as a likely vaccine candidate. This however, needs to be evaluated further. More importantly the comparison with BCG in terms of providing protection

在探究了Rv1507A的免疫反应以及Ms_Rv1507A在巨噬细胞中持久性增强与应激反应相关性的基础上，我们通过气溶胶途径评估其效果以模拟实际感染。气溶胶感染后肺组织的病理学分析显示，与载体对照组$(\mathrm{Ms}_\mathrm{Vc})$相比，感染Ms_Rv1507A的动物肺实质中淋巴细胞浸润增强（图8）。气管内接种后未发现任何肉芽肿样结构或干酪样坏死病变。这些观察结果表明，Ms_Rv1507A比Ms_Vc更具免疫原性，但毒力不足以引起任何不良病理变化。所有这些观察结果都表明Rv1507A可能作为疫苗候选分子，但尚需进一步评估。更重要的是，需要与卡介苗(BCG)在提供保护力方面进行比较

FIGURE 5 | M. tb Rv1507A protein elicits IgG response in immunized mice and exhibits immuno reactivity in sera of Tuberculosis patient. BALB/c mice were immunized with either purified M. tb Rv1507A protein or infected with Rv1507A knock-in M. smegmatis (Ms_Rv1507A) and control M. smegmatis (Ms_Vc). The level of IgG in the mouse sera was tested using ELISA and measured spec tro photometric ally (492nm) (A). Sera were collected from pulmonary TB patients $m=31,$ or control (Continued)

图 5 | 结核分枝杆菌 Rv1507A 蛋白在免疫小鼠中引发 IgG 反应并在结核病患者血清中显示免疫反应性。BALB/c 小鼠分别用纯化的结核分枝杆菌 Rv1507A 蛋白免疫或感染 Rv1507A 敲入耻垢分枝杆菌 (Ms_Rv1507A) 及对照耻垢分枝杆菌 (Ms_Vc)。采用 ELISA 检测小鼠血清 IgG 水平并通过分光光度法 (492nm) 测定 (A)。从肺结核患者 (n=31) 或对照组采集血清 (续)

FIGURE 5 | volunteers $m=19.$ ). Immuno reactivity of human sera against Rv1507A was tested as described in methods and measured spec tro photometric ally (450 nm) (B). Receiver operating characteristics (ROC) curve to assess the cut-off value of ELISA depicting $%$ sensitivity and $%$ specificity (C). Data are shown as mean $\pm$ SEM. Statistical significance was determined with one tailed Mann–Whitney test. $p<0.05$ was considered significant. $^{\star\star}p<0.01$ , $^{\star\star\star}p<0.0001$ .

图 5 | 志愿者 $m=19.$ )。人类血清对 Rv1507A 的免疫反应性按方法描述进行测试，并通过分光光度法 (450 nm) 测量 (B)。受试者工作特征 (ROC) 曲线用于评估 ELISA 的临界值，显示 $%$ 敏感性和 $%$ 特异性 (C)。数据以均值 $\pm$ 标准误表示。统计学显著性通过单尾 Mann-Whitney 检验确定。$p<0.05$ 视为显著。$^{\star\star}p<0.01$，$^{\star\star\star}p<0.0001$。

against M. tb infection needs to be considered to validate its vaccine efficacy.

验证其疫苗效力时需考虑抗结核分枝杆菌（M. tb）感染的效果。

DISCUSSION

讨论

Given the goal to eradicate TB by 2035 (3), it is imperative to embark on parallel strategies based on new drugs, repurposed drugs, and novel vaccines with emphasis on route of administration to generate protective immunity (7, 57, 58). In this study, we evaluated the pro-host and possible vaccine potential of antigenic protein Rv1507A, which is absent in BCG strain. In depth in-silico analysis of Rv1507A showed that the protein is enriched with alpha helix and beta sheets with a disordered domain in C-terminal region. These disordered regions provide structural plasticity and conformation al adaptability to proteins to enhance their binding promiscuity with various ligands. This could help pathogens to compensate for the genome reduction due to reductive evolution (59) by resorting to disordered proteins, moonlighting functions and protein promiscuity (45, 60, 61). In-silico analysis for epitope prediction and antigen i city revealed that this protein is highly antigenic. Rv1507A, being unique for M. tb and especially absent in BCG (Data in communication), could also be a potential immuno diagnostic biomarker for tuberculosis. This specificity toward pathogens reveals that these pathogen specific proteins must be necessary for survival inside host and might have been lost through reductive evolution by non-pathogens (59). These proteins are thus at the forefront of host pathogen interaction and need to be explored for their possible use as diagnostics or as vaccine candidates.

鉴于到2035年消灭结核病的目标(3)，必须基于新药、老药新用和新型疫苗并行推进策略，并重点关注给药途径以产生保护性免疫(7, 57, 58)。本研究评估了BCG菌株中缺失的抗原蛋白Rv1507A的宿主促进作用及其潜在疫苗价值。对Rv1507A的深度计算机模拟分析显示，该蛋白富含α螺旋和β折叠，其C端区域存在无序结构域。这些无序区域通过结构可塑性和构象适应性增强了蛋白与多种配体的混杂结合能力。这种特性可能帮助病原体通过无序蛋白、兼职功能和蛋白混杂性(45, 60, 61)来补偿因还原进化导致的基因组缩减(59)。表位预测和抗原性的计算机模拟分析表明该蛋白具有高度抗原性。由于Rv1507A是结核分枝杆菌特有且BCG中缺失的蛋白(数据交流中)，它还可能成为结核病的潜在免疫诊断生物标志物。这种病原体特异性表明，这些蛋白对宿主内生存至关重要，非病原体可能通过还原进化丢失了这些蛋白(59)。因此这些蛋白处于宿主-病原体相互作用的前沿，需要探索其作为诊断标志物或候选疫苗的应用潜力。

Macrophages, being the sentinels of the immune system, are the primary cells that encounter pathogen. M. tb has developed different strategies to invade and adapt inside macrophages and dendritic cells that form the primary defense against infections (62). Macrophages and dendritic cells are antigen-presenting cells that modulate function of other cells through cytokines. Cell-mediated immunity governed by cytokines such as IFN $\gamma$ and TNF $\alpha$ are mainly responsible to inhibit the growth of $M$ . tb and clear the infection (63). Previous studies on mice which were knocked down for pro-inflammatory genes like IFN $\gamma$ and TNF $\alpha$ showed these to be more prone to M. tb infection (64–66). The pro-inflammatory cytokine milieu generates Th1 protective response that allows efficient clearance of bacteria. The role of IL-6 cytokine is controversial as it has been found to have both protective and pathological roles in TB infection. During early phase of infection, it is involved in protection; while in chronic phase of TB infection, it is associated with pathology (67). Evaluation of IL-6 levels from the collected super nat ant from RAW264.7 cells exhibited dose dependent increase. Treatment of Rv1507A also activates innate immunity by activating macrophages that is evident by enhanced expression of co-sti mula tory markers on these professional APCs. CD80 and CD86 provide co-sti mula tory signal to T cells via CD28 during antigen presentation and are responsible for effective activation of $\mathrm{T}$ cells for proliferation and cytokine production (68, 69). CD40 is another co-sti mula tory molecule that activates APCs upon interaction with CD154 (CD40L) on $\mathrm{CD4T}$ cells (70). This activation translates into effective micro bic i dal response by these APCs through NO and ROS that also modulate initiation and progression of cellular and humoral adaptive immunity. CD69 is an early activation marker of T cells and can be induced on other cells of he ma top oie tic lineage. Enhanced expression of these molecules suggests presence of activated T cells that indicates effective T cell immunogen i city that corroborates in-silico data for T cell epitope prediction. This activation of innate receptors generates antimicrobial response apart from effective activation of adaptive arm of immunity (Figure 9).

巨噬细胞作为免疫系统的哨兵，是遭遇病原体的首要细胞。结核分枝杆菌(M. tb)已进化出多种策略入侵并适应巨噬细胞和树突状细胞——这些构成抗感染第一道防线的细胞[62]。作为抗原呈递细胞，巨噬细胞和树突状细胞通过细胞因子调控其他细胞功能。由IFN$\gamma$和TNF$\alpha$等细胞因子主导的细胞免疫应答是抑制结核杆菌生长并清除感染的主要机制[63]。既往对IFN$\gamma$和TNF$\alpha$等促炎基因敲除小鼠的研究表明，这些小鼠更易感染结核杆菌[64-66]。促炎性细胞因子环境可激发Th1保护性应答，从而实现高效清除细菌。IL-6细胞因子的作用存在争议，研究发现其在结核感染中兼具保护性和病理性作用：感染早期发挥保护功能，而在结核慢性感染期则与病理损伤相关[67]。

对RAW264.7细胞上清液中IL-6水平的检测显示其呈剂量依赖性升高。Rv1507A蛋白处理还能通过激活巨噬细胞来启动先天免疫，这些专职抗原呈递细胞表面共刺激分子表达增强即为明证。CD80和CD86在抗原呈递过程中通过CD28向T细胞传递共刺激信号，这对有效激活T细胞增殖及细胞因子分泌至关重要[68,69]。CD40是另一种共刺激分子，当其与CD4T细胞表面的CD154(CD40L)结合时可激活抗原呈递细胞[70]。这种激活会促使抗原呈递细胞通过一氧化氮(NO)和活性氧簇(ROS)产生有效的杀菌应答，同时调控细胞免疫与体液免疫的启动与进程。CD69作为T细胞早期活化标志物，也可在其他造血谱系细胞中诱导表达。这些分子表达增强提示活化T细胞的存在，表明有效的T细胞免疫原性，这与T细胞表位预测的计算机模拟数据相互印证。先天免疫受体的激活不仅能有效启动适应性免疫，还可产生直接的抗菌应答(图9)。

Adaptive immune cells like $\mathrm{CD4^{+}}$ and $\mathrm{CD}8^{+}$ T cells modulate the function of other immune cells that in turn maintain checks and balances on $M$ . tb infection (71, 72). $\mathrm{CD4^{+}}$ and $\mathrm{CD}8^{+}$ T cells are important during acute phase of infection and in clearance of chronic infection, respectively (73, 74). Moreover, cytotoxic $\mathrm{CD}8^{+}$ T cells play an important role, through MHC I pathway, in protection against intracellular pathogens. $\mathrm{CD4^{+}}$ T cells on the other hand play a crucial role in response to antigen processed by APCs through MHC II pathway. These cells differentiate into different effector subtypes and modulate the cytokine profile which leads to cellular proliferation of B cells and activation of antigen presenting cells (APCs). The classical approach to estimate the vaccine efficacy of a candidate protein is to evoke Th1 response that is usually considered protective in case of intracellular infections. The hallmark Th1 cytokine IFN $\gamma$ activates macrophages for pathogen clearance and TNF acts in synergy to produce NO to unleash b acte rio static effect of macrophages. TNF also modulates migratory potential and thus leads to granuloma formation to ward off the bacteria from further transmission (23). We observed induction of proinflammatory cytokine $(\mathrm{IFN}{-\gamma}$ ) in immunized mice, which clearly depicts pro-host immune modulatory effect of the antigenic protein (Figure S5). This immune effect was devoid of any bias as we avoided use of any adjuvant due to their inherent ability to skew resultant immune response toward Th1 or Th2 depending on their ph y sio chemical properties (47, 48, 75, 76). The adjuvants are required to activate innate immune receptors, such as pattern recognition receptors (PRRs), toll-like receptors (TLRs), nucleotide-binding oli gomer is ation domain (NOD)-like receptors, or retinoic acid-inducible gene-I (RIGI)-like receptors, each of which initiate different downstream cytokine signaling to mediate protection against pathogens. Treatment of macrophages with mycobacteria l proteins reveals their self-adjuvant properties as evident from up-regulation of

适应性免疫细胞如 $\mathrm{CD4^{+}}$ 和 $\mathrm{CD8^{+}}$ T 细胞通过调控其他免疫细胞功能，进而维持对结核分枝杆菌 (M. tb) 感染的动态平衡 (71, 72)。$\mathrm{CD4^{+}}$ T 细胞在感染急性期发挥关键作用，而 $\mathrm{CD8^{+}}$ T 细胞则主要参与慢性感染的清除过程 (73, 74)。此外，细胞毒性 $\mathrm{CD8^{+}}$ T 细胞通过 MHC I 类分子途径，在抵御胞内病原体感染中具有重要作用。$\mathrm{CD4^{+}}$ T 细胞则通过 MHC II 类分子途径响应抗原呈递细胞 (APCs) 处理的抗原，分化为不同效应亚型并调控细胞因子谱，从而促进 B 细胞增殖和抗原呈递细胞活化。评估候选蛋白疫苗效力的经典方法是诱导 Th1 型免疫应答，该应答通常对胞内感染具有保护作用。标志性 Th1 细胞因子 IFN-$\gamma$ 可激活巨噬细胞清除病原体，TNF-$\alpha$ 则通过协同作用产生一氧化氮 (NO)，释放巨噬细胞的抑菌效应。TNF-$\alpha$ 还能调节迁移潜能，促进肉芽肿形成以阻断细菌传播 (23)。我们在免疫小鼠中观察到促炎细胞因子 $(\mathrm{IFN}{-\gamma})$ 的诱导表达，明确揭示了抗原蛋白的促宿主免疫调节效应 (图 S5)。该免疫效应不存在偏好性，因为我们未使用任何佐剂——佐剂会因其理化特性固有地使免疫应答偏向 Th1 或 Th2 型 (47, 48, 75, 76)。佐剂通常用于激活先天免疫受体（如模式识别受体 (PRRs)、Toll 样受体 (TLRs)、核苷酸结合寡聚化结构域 (NOD) 样受体或视黄酸诱导基因 I (RIGI) 样受体），这些受体会启动不同的下游细胞因子信号通路以介导抗病原体保护。用分枝杆菌蛋白处理巨噬细胞可观察到其自身佐剂特性，这体现于...

FIGURE 6 | Rv1507A knock-in M. smegmatis exhibits enhanced uptake by macrophage and induction of stress response. RAW 264.7 macrophage cells infected with fluorescent SYTO-9 stained Ms_Vc and Ms_Rv1507A. The uptake of Ms_Vc and Ms_Rv1507A within the cells were visualized microscopically (magnification 100X, scale bar represents $5\mathrm{m}$ (A) and by CFU assay (B). RAW 264.7 cells infected with Ms_Vc (black box) and Ms_1507A (gray box) were stained with Cell ROX (Continued)

图 6 | 敲入 Rv1507A 的耻垢分枝杆菌表现出增强的巨噬细胞摄取和应激反应诱导能力。用荧光染料 SYTO-9 标记的 Ms_Vc 和 Ms_Rv1507A 感染 RAW 264.7 巨噬细胞，通过显微镜观察细胞内对 Ms_Vc 和 Ms_Rv1507A 的摄取情况 (放大倍数 100X，比例尺为 $5\mathrm{m}$ ) (A)，并通过 CFU 实验进行验证 (B)。感染 Ms_Vc (黑色方框) 和 Ms_1507A (灰色方框) 的 RAW 264.7 细胞用 Cell ROX 染色 (待续)

FIGURE 6 | Orange and assessed for reactive oxygen species (ROS). Representative data shows the mean fluorescence intensity (MFI) of ROS generated within the macrophage as mean $\pm$ SEM from three separate experiments (C). Statistical significance was determined by two-way ANOVA. Cell culture super nat ants were assessed for levels of Nitric Oxide $(\mathsf{N O})$ , using Griess reagent assay, after 12, 24, and $48\mathsf{h}$ post infection of RAW264.7 cells with Ms_Rv1507A (gray box) or Ms_Vc (black box). Representative data show the concentration of NO as mean $\pm$ SEM from three separate experiments (D). Statistical significance was determined by two-way ANOVA. RAW264.7 cells were cultured in presence of Ms_Vc or Ms_Rv1507A at an MOI of 10:1. After $48\mathsf{h}$ , cells were stained with AnnexinV-7AAD dye and apoptosis was assessed. Representative data show the percent apoptotic cells as mean $\pm$ SEM from three separate experiments. Cells treated with St a uro spor in $(500\mathsf{n M})$ were used as a positive control $(\mathsf{P C})$ (E). Statistical significance was determined by one-way ANOVA. $p<0.05$ was considered significant, $^{\star}p<0.05$ , $^{\star\star}p<$ 0.01, $^{\star\star\star}p<0.001$ , and $^{\star\star\star}p<0.0001$ .

图 6 | 检测活性氧 (ROS) 的橙染结果。代表性数据显示巨噬细胞内生成的 ROS 平均荧光强度 (MFI) 为均值 $\pm$ 标准误 (SEM) (C)，数据来自三次独立实验。统计学显著性采用双向方差分析判定。分别用 Ms_Rv1507A (灰色框) 和 Ms_Vc (黑色框) 感染 RAW264.7 细胞后，在 12、24 和 $48\mathsf{h}$ 时使用 Griess 试剂法检测细胞培养上清液中一氧化氮 $(\mathsf{N O})$ 水平。代表性数据显示 NO 浓度为均值 $\pm$ SEM (D)，数据来自三次独立实验。统计学显著性采用双向方差分析判定。RAW264.7 细胞以 10:1 的感染复数 (MOI) 与 Ms_Vc 或 Ms_Rv1507A 共培养 $48\mathsf{h}$ 后，用 AnnexinV-7AAD 染色评估细胞凋亡。代表性数据显示凋亡细胞百分比为均值 $\pm$ SEM (E)，数据来自三次独立实验。以 Staurosporine $(500\mathsf{n M})$ 处理的细胞作为阳性对照 $(\mathsf{P C})$。统计学显著性采用单因素方差分析判定。$p<0.05$ 视为显著，$^{\star}p<0.05$，$^{\star\star}p<0.01$，$^{\star\star\star}p<0.001$，$^{\star\star\star}p<0.0001$。

various co-sti mula tory receptors. Adjuvant role of mycobacteria l proteins has been earlier delineated and our observations also reflect the same (77, 78). The secretion of TNF and $\operatorname{IFN-}\gamma$ were found to be significant from $\mathrm{CD4^{+}}$ as well as $\mathrm{CD}8^{+}$ T cells of Rv1507A sensitized mice as compared to control. Even though our observations also suggest polyclonal activation by antigenic stimulation, as revealed by most cells showing secretion, there was a distinct antigen induced increase in cells secreting both TNFα and IFNγ upon immunization. These polyfunctional T cells are one of the classical predictors of protective immunity and act as a signature of vaccine induced protection (79). It is conceivable that cells expressing multiple effector molecules would be more effective to control pathogens as these cytokines act synergistic ally. Various other M. tb antigens, which form the components of vaccines in clinical trials, have been shown to elicit poly-functional $\mathrm{CD4^{+}}$ T cells (79). The other functional attributes of these poly functional cells include their enhanced potency for cytokine secretion and long-lived memory function with ability to migrate into lungs. We also analyzed the memory like phenotype of T cells after Rv1507A immunization as well as after infection with Ms_Rv1507A. $\mathrm{T_{EM}}$ are involved in immediate responses and induce cytolysis of infected cells. These cells are devoid of homing receptors thus are usually present in blood or peripheral tissues. TCM on other hand are highly pro life rat ive with ability to generate effector cells. These cells have longer life span and are involved in long term protection (80).

多种共刺激受体。分枝杆菌蛋白的佐剂作用先前已有阐述，我们的观察结果也反映了这一点 (77, 78)。与对照组相比，Rv1507A致敏小鼠的CD4+和CD8+ T细胞分泌TNF与$\operatorname{IFN-}\gamma$的水平显著升高。尽管我们的观察也提示抗原刺激引起的多克隆激活（表现为多数细胞分泌$\mathrm{TNF}$），但免疫后同时分泌TNFα和IFNγ的细胞数量出现明显的抗原诱导性增加。这类多功能T细胞是保护性免疫的经典预测指标之一，可作为疫苗诱导保护的标志 (79)。可以推测，表达多种效应分子的细胞能通过细胞因子的协同作用更有效地控制病原体。其他多种作为临床试验疫苗组分的M.tb抗原也已被证明可激发多功能的$\mathrm{CD4^{+}}$ T细胞 (79)。这些多功能细胞的其他功能特征包括更强的细胞因子分泌能力、长效记忆功能以及向肺部迁移的能力。我们还分析了Rv1507A免疫后及Ms_Rv1507A感染后T细胞的记忆样表型。$\mathrm{T_{EM}}$参与即时反应并诱导感染细胞的细胞溶解，这些细胞缺乏归巢受体，因此通常存在于血液或外周组织中。而TCM则具有高度增殖能力，可生成效应细胞，这些细胞寿命更长，参与长期保护 (80)。

Apart from cell mediated immunity, Rv1507A also evoked humoral immunity in mice as revealed by increased levels of protein specific antibodies in immunized animals. Although the role of antibodies in protection against TB has been subjugated by cell mediated immunity however recent evidence suggests that antibodies can positively modulate the immune responses against M. tb. It is evident that multifaceted B cells, by virtue of antigen presentation, antibody and cytokine production, are able to exert significant effect on T cell mediated immunity that is classically considered as critical for TB control (81– 83). Extrapolating these findings to human host we expected antibodies against Rv1507A in sera of TB patients. A clear nonoverlap in IgG reactivity in PTB patients and healthy controls points to the fact that response by Rv1507A is highly specific and attests our observation that Rv1507A is absent in mycobacteria except M. tb. The credibility of a diagnostic test is usually measured by AUC of an ROC curve. The AUC value of 0.996 depicts excellent diagnostic utility of this protein. Likelihood ratio (LR) of a test is also an important statistical method to better evaluate the diagnostic test. In our study, LR of positive test values was very high with lower corresponding LR negative values (Figure S9). The test also determined that $98%$ of samples were correctly classified at the selected cut-off value that yielded maximum sensitivity and specificity (Figure S9). The sero-specificity of Rv1507A further suggests that it can be a suitable pathogen specific marker for diagnosis of active TB that can be used in tandem with host specific marker such as resistin (84–86) to design bimodal, rapid, and simple point of care (POC) diagnostic tests with enhanced ability to detect disease at early stages.

除了细胞介导的免疫反应外，Rv1507A还通过提高免疫动物体内蛋白特异性抗体水平，激发了小鼠的体液免疫。尽管抗体在结核病防护中的作用长期被细胞免疫主导，但最新证据表明抗体能积极调控针对结核分枝杆菌的免疫应答。显然，多功能B细胞通过抗原呈递、抗体及细胞因子分泌，能显著影响传统上被视为结核病防控关键的T细胞免疫[81-83]。将这些发现类推至人类宿主，我们预期结核患者血清中存在抗Rv1507A抗体。肺结核患者与健康对照组的IgG反应性明显无重叠，表明Rv1507A的应答具有高度特异性，并证实了该蛋白仅存在于结核分枝杆菌中的观察结果。诊断试验的可信度通常通过ROC曲线的AUC值衡量，0.996的AUC值表明该蛋白具有极佳诊断效能。似然比(LR)是评估诊断试验的另一重要统计方法，本研究中阳性检测的LR值极高而阴性LR值较低(图S9)。该试验还确定在选定截断值下，98%的样本能被正确分类，此时灵敏度与特异性达到最大化(图S9)。Rv1507A的血清特异性进一步提示，它可作为诊断活动性结核的病原体特异性标志物，与抵抗素(84-86)等宿主标志物联用，设计兼具双模态、快速、简便的床旁诊断方案，提升早期疾病检出能力。

In line with these observations, we expected increased virulence in M. smegmatis when supplemented with Rv1507A. Our results suggested that Rv1507A knock-in M. smegmatis (Ms_Rv1507A) can survive for prolonged duration within macrophage. In response to infection by mycobacterium, macrophages mount physiological responses either in form of generation of ROS and reactive nitrogen species (RNS) to get rid of pathogen (87) or may undergo apoptosis in case it fails to get rid of pathogen (88). These results provide a mechanistic basis of induction of apoptosis in macrophages, infected with recombinant Ms_Rv1507A. Increased levels of ROS and NO leads to stress within the macrophages that result in apoptosis of macrophages. While cells undergo apoptosis to suppress replication of bacteria, pathogens such as M. tb try to inhibit apoptosis as a mechanism to subdue host immune response and can alternatively induce necrosis to disseminate infection to surrounding cells (89). Virulence and cellular apoptosis are two different aspects employed by bacteria for pathogen es is. While M. tb induces necrosis for dissemination, BCG and attenuated mutants including $\mathrm{H}_{37}\mathrm{Ra}$ predominantly induce apoptosis. Innate control of early bacterial growth is mediated through induction of apoptosis in infected macrophages which helps in dissemination of antigen reservoir from the cell. These antigens released from apoptotic cells allow cross priming in dendritic cells and activation of T cell mediated acquired immunity (90, 91). Apoptosis is considered as a host defense mechanism as it allows sequestration of pathogens within apoptotic vesicles leading to decreased bacterial viability (92).

与这些观察结果一致，我们预期在补充Rv1507A后，耻垢分枝杆菌(M. smegmatis)的毒力会增强。结果表明，Rv1507A敲入的耻垢分枝杆菌(Ms_Rv1507A)能在巨噬细胞内长期存活。针对分枝杆菌感染，巨噬细胞会通过产生活性氧(ROS)和活性氮物种(RNS)来清除病原体[87]，或在清除失败时发生凋亡[88]。这些结果揭示了重组Ms_Rv1507A感染诱导巨噬细胞凋亡的机制基础。ROS和一氧化氮(NO)水平升高会导致巨噬细胞内应激，最终引发凋亡。虽然细胞通过凋亡来抑制细菌复制，但结核分枝杆菌(M. tb)等病原体会抑制凋亡以削弱宿主免疫应答，并可能转而诱导坏死来向周围细胞传播感染[89]。毒力与细胞凋亡是细菌致病机制的两个不同方面：结核分枝杆菌通过诱导坏死实现传播，而卡介苗(BCG)和减毒突变株(如$\mathrm{H}_{37}\mathrm{Ra}$)主要诱导凋亡。早期细菌生长的先天控制通过感染巨噬细胞的凋亡实现，这有助于细胞释放抗原库。凋亡细胞释放的抗原能促进树突状细胞的交叉呈递，激活T细胞介导的获得性免疫[90,91]。凋亡被视为宿主防御机制，因其能将病原体隔离在凋亡小泡内从而降低细菌存活率[92]。

In conclusion, we have shown the immunogen ic and antigenic potential of M. tb specific Rv1507A protein that is absent in BCG vaccine. It elicits pro-host immune response under in-vitro or in-vivo conditions and can be further evaluated as a possible standalone subunit vaccine candidate. Though subunit vaccines are relatively safer than live vaccines but low immunogen i city and inadequacy of long-lived protection are areas of concern. The laborious process of purifying vaccine grade antigen multiple times and the use of adjuvant for enhanced immunogen i city that can potentially skew the immune responses are other potential limitations. Interestingly Rv1507A was inherently immunogen ic, thus despite avoiding the use of adjuvant, it evoked substantial innate and adaptive immune response. On the contrary, higher magnitude of immune and memory response induced by Ms_Rv1507A compared to that of recombinant protein points to likely better efficacy of live vaccines. Live vaccines are highly immunogen ic, and a single dose is usually enough for long term protection, but reversion to virulence is a serious limitation. Aerosol infection with Ms_Rv1507A reiterated that this recombinant vaccine is immunogen ic but not virulent to cause any pathology. Further, the revival of BCG as a vaccine due to recent advancements in understanding of vaccine induced protection and the emerging role of trained immunity advocates use of Rv1507A for production of recombinant BCG vaccine. Integration of Rv1507A in BCG can elicit reinvigorate the missing central memory compartment that could sustain long term protection, missing in BCG. A similar study has already exhibited that RD 4 (Region of Difference 4) (Rv1501-1508c) that encompasses Rv1507A, when incorporated into BCG, demonstrated improved protection of zebra fish against M. marinum challenge than the parental BCG (93). Therefore, this pro-host antigenic protein could be a probable candidate to produce recombinant BCG with enhanced immune response. A lot of progress has been made to engineer BCG that has led to development of promising vaccines, however still considerable efforts are required to generate vaccines with functional long lasting memory.

综上所述，我们证实了BCG疫苗中缺失的结核分枝杆菌特异性Rv1507A蛋白具有免疫原性和抗原潜力。该蛋白在体外或体内条件下均能激发促宿主免疫反应，可作为潜在的独立亚单位疫苗候选分子进行进一步评估。虽然亚单位疫苗比活疫苗更安全，但其免疫原性较弱且缺乏长效保护仍是关注焦点。此外，多次纯化疫苗级抗原的繁琐工序，以及为增强免疫原性使用佐剂可能导致的免疫应答偏移，都是潜在限制因素。值得注意的是，Rv1507A具有天然免疫原性，即使不使用佐剂也能激发显著的先天性和适应性免疫应答。相比之下，Ms_Rv1507A诱导的免疫与记忆反应强度高于重组蛋白，表明活疫苗可能具有更优效力。活疫苗免疫原性极强，单次接种通常即可实现长期保护，但毒力返祖是其重大缺陷。Ms_Rv1507A气溶胶感染实验再次证实该重组疫苗具有免疫原性但不会引发病理损伤。随着对疫苗诱导保护机制认识的深入及训练免疫新概念的兴起，BCG作为疫苗的价值被重新发掘，这支持将Rv1507A用于重组BCG疫苗开发。将Rv1507A整合至BCG可激活缺失的中心记忆区室，弥补BCG缺乏长期保护的缺陷。已有研究表明，包含Rv1507A的RD4区（Rv1501-1508c）整合至BCG后，相比亲本BCG能更有效保护斑马鱼抵抗海分枝杆菌攻击 [93]。因此，这种促宿主抗原蛋白有望成为开发具有增强免疫应答的重组BCG疫苗的候选分子。尽管BCG改造已取得重大进展并催生多个前景良好的疫苗，但要开发具有功能性长效记忆的疫苗仍需付出巨大努力。

FIGURE 7 | Increased sur viv ability of Ms_Rv1507A within macrophages and resistance to stress induction in culture. RAW 264.7 cells were co-cultured with Ms_Vc or Ms_Rv1507A and the colony forming units/ml were counted after 12 h, $^{24\mathsf{h}}$ , and $48\mathsf{h}$ . Representative data from three experiments show survival of viable Ms_Vc (black box) and Ms_Rv1507A (gray box) as mean±SEM (A). Ms_Vc and Ms_Rv1507A cells were cultured in presence of 5 and $10\mathsf{m M}$ of sodium nitrite. The cell viability was assessed at 3 h, 6 h, and 9 h. Representative data from three experiments show the survival in terms of log10 CFU/ml as mean±SEM (B). Ms_Vc and Ms_Rv1507A cells were cultured in presence of 5 and $10\mathsf{m M}$ of hydrogen peroxide. The cell viability was assessed at 3, 6, and 9 h. Representative data from three experiments show the survival in terms of $\log_{10}$ CFU/ml as mean $\pm$ SEM (C). Statistical significance was determined by two-way ANOVA. $p<0.05$ was considered significant, $^{\star}p<0.05$ , $^{\star\star}p<0.01$ , $^{\star\star\star}p<0.001$ , and $^{\star\star\star}p<0.0001$ .

图 7 | Ms_Rv1507A在巨噬细胞内生存能力增强及培养条件下对胁迫诱导的抗性。RAW 264.7细胞与Ms_Vc或Ms_Rv1507A共培养12小时、$^{24\mathsf{h}}$和$48\mathsf{h}$后统计菌落形成单位/毫升。三次实验的代表性数据显示存活Ms_Vc(黑框)与Ms_Rv1507A(灰框)的平均值±SEM(A)。Ms_Vc和Ms_Rv1507A细胞在5及$10\mathsf{m M}$亚硝酸钠条件下培养，分别于3、6、9小时检测细胞活性。三次实验的代表性数据以log10 CFU/ml表示存活率均值±SEM(B)。Ms_Vc和Ms_Rv1507A细胞在5及$10\mathsf{m M}$过氧化氢条件下培养，分别于3、6、9小时检测细胞活性。三次实验的代表性数据以$\log_{10}$ CFU/ml表示存活率均值$\pm$SEM(C)。统计学显著性采用双因素方差分析判定，$p<0.05$视为显著，$^{\star}p<0.05$，$^{\star\star}p<0.01$，$^{\star\star\star}p<0.001$，$^{\star\star\star}p<0.0001$。

FIGURE 8 | Ms_Rv1507A increases the infiltration of lymphocytes in the lung tissue of immunized mice. Lungs were recovered after 30 days from BALB/c mice $(n=6)$ that were injected with either Phosphate buffer saline (un-infected) or Ms_Vc $(1\times10^{7})$ or Ms_Rv1507A cells $(1\times10^{7})$ intra tracheal ly. Lungs were washed in PBS and then fixed in $10%$ formalin solution. After fixation, lungs were fine–sectioned and stained with He mato xy lin and Eosin (HE) solution. Eosin is pink and stains proteins non-specifically. In a typical tissue, nuclei are stained blue, whereas the cytoplasm and extracellular matrix have varying degrees of pink staining. The images were captured for at least 5 different fields in 6 mice.

图 8 | Ms_Rv1507A 增强免疫小鼠肺组织中淋巴细胞浸润。BALB/c 小鼠 $(n=6)$ 气管内注射磷酸盐缓冲液 (未感染组)、Ms_Vc $(1\times10^{7})$ 或 Ms_Rv1507A 细胞 $(1\times10^{7})$ 30天后取肺组织。肺组织经 PBS 冲洗后固定于 $10%$ 福尔马林溶液，精细切片并进行苏木精-伊红 (HE) 染色。伊红呈粉红色非特异性染色蛋白质，典型组织中细胞核呈蓝色，而细胞质和细胞外基质呈现不同程度的粉红色染色。每组6只小鼠至少拍摄5个不同视野的图像。

FIGURE 9 | Model depicting the immuno modulator y role of Rv1507A. (A) Recombinant M. smegmatis expressing Rv1507A is ph ago cyto sed by macrophage which increases generation of ROS and NO that leads to induction of apoptosis. Macrophage exposed to Rv1507A secretes pro-inflammatory cytokines such as TNF $\cdot\alpha$ , IL-12, and IL-6. It also leads to increase in the expression of MHCI, MHCII, CD40, CD80, and CD86 that indicates efficient antigen presentation and co-stimulation. (B) Immunization with Rv1507A protein or Ms_Rv1507A in mice leads to increased humoral and cell mediated immunity. A robust effector memory $(\mathsf{T}{\mathsf{E M}})$ and central memory $(\top_{\mathsf{C M}})$ response is also evoked along with enhanced poly-functional $\mathtt{C D4^{+}}$ as well as $\mathtt{C D8^{+}}$ cells.

图 9 | Rv1507A免疫调节作用模型。(A) 表达Rv1507A的重组耻垢分枝杆菌被巨噬细胞吞噬后，增加ROS和NO的产生，从而诱导细胞凋亡。暴露于Rv1507A的巨噬细胞分泌促炎细胞因子如TNF-α、IL-12和IL-6。同时导致MHCI、MHCII、CD40、CD80和CD86表达增加，表明有效的抗原呈递和共刺激作用。(B) 用Rv1507A蛋白或Ms_Rv1507A免疫小鼠可增强体液免疫和细胞免疫。同时引发强效的效应记忆(TEM)和中枢记忆(TCM)反应，并增强多功能性CD4+和CD8+细胞。