王建忠#, 赵建伟#, 王春凤
吉林农业大学动物科学技术学院, 吉林省动物微生态制剂工程研究中心, 吉林 长春 130118
收稿日期：2018-04-24；修回日期：2018-07-19；网络出版日期：2018-11-27
基金项目：吉林省青年人才托举工程（201709）；吉林农业大学科研启动基金（201703）
*通信作者：王春凤, Tel/Fax: +86-431-84533425; E-mail: wangchunfeng@jlau.edu.cn
#并列第一作者
摘要：细菌样颗粒（Bacterium-like particles，BLPs）是一种新型非遗传修饰型乳酸菌表面展示技术，外源蛋白可通过锚钩蛋白锚定于经热酸处理而得的乳酸菌肽聚糖骨架表面，形成空心表面展示颗粒。因其安全性高、表面展示密度大、黏膜递送效率高，又兼有佐剂效应，BLPs广泛应用于黏膜疫苗和黏膜佐剂的开发、病毒抗原的纯化、生物催化剂的制备等领域。本文就BLPs的构建、独特优势、目前的应用及尚需解决的问题等方面进行详细综述，以期展现BLPs新型表面展示平台的广阔应用前景。
关键词：细菌样颗粒表面展示技术乳酸菌
Bacterium-like particles—a novel surface display technology for lactic acid bacteria and its application
Jianzhong Wang#, Jianwei Zhao#, Chunfeng Wang
Jilin Provincial Engineering Research Center of Animal Probiotics, College of Animal Science and Technology, Jilin Agricultural University, Changchun 130118, Jilin Province, China
Received 24 April 2018; Revised 19 July 2018; Published online 27 November 2018
*Corresponding author: Chunfeng Wang, Tel/Fax: +86-431-84533425; E-mail: wangchunfeng@jlau.edu.cn
Supported by the Youth Talent Promotion Project of Jilin Province (201709) and by the Doctoral Project Sponsored by the Scientific Research Foundation of Jilin Agricultural University (201703)
#These authors contributed equally to this work
Abstract: The studies on lactic acid bacteria as live vehicles for expression and display of heterologous proteins or antigens have gained great progress in the past decades. Recently, a novel display system called Bacterium-like particles was designed and described. This system is based on nonliving and non-genetically modified gram-positive bacterial cells, generally the innocuous bacterium Lactococcus lactis pretreated by hot acids. The peptidoglycan-binding domain of lactococcal AcmA protein has been used as the protein anchor for heterologous surface display of various proteins on lactic acid bacteria. Compared to the living lactic acid bacteria, Bacterium-like particles have a higher binding capacity, safety, delivering efficiency, and less anticarrier response. They have been widely used in the development of mucosal vaccines and adjuvants, purification of viral antigens, and preparation of biocatalysts. In this review, we focus on the construction, unique advantages of Bacterium-like particles, and successful application in many fields. Finally, we will discuss the broad application prospects and problems to be solved in the nearly future.
Keywords: Bacterium-like particlessurface display techniquelactic acid bacteria
乳酸菌(lactic acid bacteria，LAB)是一类可以将碳水化合物发酵成乳酸的革兰氏阳性菌的总称。包含乳酸杆菌属、乳酸球菌属、链球菌属、肠球菌属、明串珠菌属、片球菌属和双歧杆菌属等。乳酸菌是公认安全(generally regarded as safe，GRAS)的食品级微生物，在过去20多年里，重组乳酸菌作为抗原蛋白、药物分子、外源DNA的黏膜递送工具，广泛应用于疫苗研制、药物递送、基因治疗等各个领域[1]。乳酸菌作为递送载体外源蛋白有3种表达方式：一是外源蛋白表达于乳酸菌细胞内；二是在信号肽的作用下将外源蛋白分泌到细胞外；三是将外源蛋白表达后锚定到菌体表面[2]，其免疫效果以表面锚定者最佳。目前为止，一系列病毒、细菌和寄生虫抗原在乳酸菌表面得到了成功展示。但由于重组乳酸菌为活酶遗传修饰生物体(genetically modified organisms，GMO)，转基因和活菌的应用可能存在外源基因或修饰基因向环境扩散或横向传递给其他生物体的安全隐患[2]。2006年，Bosma等首次开发了一种基于细菌样颗粒(Bacterium-like particles，BLPs)的非活性(non-living)、非遗传修饰(non-GMO)的新型乳酸菌表面展示技术，在黏膜疫苗的研发中显示出安全、高效的独特优势[3]。
1 BLPs新型乳酸菌表面展示技术 BLPs是一种将食品级乳酸菌经热酸处理，去除原有蛋白质、核酸和脂磷壁酸等胞内外大分子物质而只留下细胞壁肽聚糖(peptidoglycan，PGN)骨架的空心颗粒，其大小和形态与活菌相似，因此被称为细菌样颗粒[4]，这一处理方法适用于所有具有厚而致密细胞壁的革兰氏阳性菌，因此，也曾被称为革兰氏阳性菌增强基质(Gram-positive enhancer matrix，GEM)，但大部分细菌在这样剧烈的化学处理下易于裂解，而乳酸菌因其良好的耐酸性则不会裂解[5]。作为新型抗原展示平台，外源抗原蛋白通过融合锚钩蛋白(protein anchor，PA)，牢牢地结合于肽聚糖骨架表面。该系统操作简单方便，在体外将PA和抗原蛋白融合表达后，加入热酸预处理的乳酸菌肽聚糖颗粒中孵育，在PA的帮助下，即可形成抗原展示颗粒[6](图 1)。
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| 图 1 BLPs疫苗构建示意图[5] Figure 1 Schematic representation illustrating the preparation of a BLPs-based vaccine[5]. |
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通常PA来自于乳酸菌自身表达的肽聚糖水解酶(AcmA)。AcmA有2个活性中心，分别为N-端水解酶活性中心和C-端细胞壁结合活性中心。在BLPs展示体系中起锚定作用的就是AcmA的C-端结构域，它由3个被异源序列间隔开的自溶素基序(lysine motif，LysM)组成，每个LysM由45个氨基酸残基组成，且高度同源，也被称为重复基序，可以特异性地以非共价键(范德华力和氢键)的形式结合在肽聚糖骨架上，其结合能力非常强，只有在SDS或者氯化锂处理时才能将锚定蛋白解离下来[7]，可在-80 ℃、5 ℃、25 ℃等不同温度条件下稳定保存2年以上[5]。PA属于少数无种属特异性的可以结合细菌细胞壁的蛋白之一，它不仅能够识别并结合于乳酸菌细胞壁，而且还能结合于其他革兰氏阳性细菌细胞壁[7]。并有研究表明PA中LysM基序数量的多少对外源蛋白结合细胞壁的能力具有重要的影响，而只有在LysM数量为3——即天然构成的结构域时，外源蛋白的锚定效率最高[8]，正如AcmA需要有3个LysM重复基序才能表现出最佳的肽聚糖结合活性和良好的酶活性[9]。
2 BLPs表面展示系统的独特优势 2.1 安全性高 与其他表面展示系统相比，乳酸菌BLPs最突出的优势为安全性高。首先，BLPs由无致病性的食品级乳酸菌制备，来源安全；其次，乳酸菌BLPs为非活性(non-living)、非遗传修饰生物体(non-GMO)，不含任何核酸物质，最大程度地减少了重组DNA向环境和其他生物传播的风险。
2.2 抗原展示密度大 BLPs可高密度地展示外源抗原蛋白，其负载能力远超越母本活菌。乳酸菌活菌表面的脂磷壁酸和磷壁酸等物质可阻碍LysM的结合，研究证实，经热酸处理去除这些物质后，其结合外源蛋白的能力可显著提高。Steen等通过免疫荧光方法检测LysM介导乳酸菌展示的外源蛋白时，发现表面有脂磷壁酸和磷壁酸等物质的位点阳性荧光斑点分布较少，而经热酸处理后阳性荧光信号显著增多，且分布于整个菌体表面[10]。Zeng等采用近场扫描光学显微技术和原子力显微镜技术分析了表面展示鼠疫杆菌V抗原细菌样颗粒的纳米结构及结合密度，即使保守计算单个颗粒表面也至少有3000个量子点，所包装的抗原分子密度为1492分子/μm2，而表达有高密度CD4分子的T细胞其密度仅为866分子/μm2[11]。另据Bosma等的计算，BLPs的饱和结合量为140-150 μg/U (2.5×109颗粒)，平均为106锚定蛋白/分子，其结合能力比依赖蛋白表达的传统重组乳酸菌载体系统高2-3个数量级[3]，能有效递送足够量的抗原物质是诱导机体产生强烈免疫反应的必要条件。
2.3 具有自体佐剂效应 Toll样受体是一类重要的模式识别受体(pattern recognition receptors，PRRs)，能通过识别不同的病原相关分子模式(pathogen-associated molecular patterns，PAMPs)，在连接固有免疫与特异性免疫的关键环节发挥着极为重要的作用[12]。BLPs的主要成分是肽聚糖，为TLR2的配体之一[13]。表达于固有免疫细胞和黏膜上皮细胞的Toll样受体(Toll-like receptors，TLRs)家族，通过与PAMPs的相互作用，启动胞内信号传导通路，激活固有免疫反应，增强天然免疫系统对病原微生物的清除和杀伤能力。现已证实BLPs通过激活TLR2信号通路，诱导宿主树突状细胞(dendritic cell，DCs)成熟，高表达CD80、CD86、CD40和MHC-Ⅱ等表面分子，成熟的DCs分泌IFN-γ、IL-2等炎性细胞因子，从而平衡Th1型和Th2型免疫，激发更有效的免疫应答反应[14-15]。相反，敲除TLR2基因的小鼠接种流感BLPs佐剂疫苗后特异性B细胞和IFN-γ分泌性T细胞数量均显著低于野生型小鼠，几乎检测不到黏膜SIgA和系统IgG2c的分泌[16]。
2.4 黏膜递送效率高 BLPs大小约1 μm，刚好是黏膜表面M细胞摄入外源抗原的理想大小，可被位于鼻咽上皮组织和肠道的M细胞有效摄入并转运至抗原递呈细胞[17-18]；同时，大小为1-2 μm的抗原颗粒可以更有效地与黏膜表面抗原递呈细胞互作促进抗原的捕获，位于肠黏膜相关淋巴组织的Peyer’s结节和鼻腔的鼻咽相关淋巴组织是机体共同黏膜免疫系统(common mucosal immune system，CMIS)的一部分，能有效诱导抗原特异性Th细胞、细胞毒性T淋巴细胞和IgA分泌B细胞反应，启动局部黏膜和全身系统性免疫应答反应[19-20]；因此，BLPs是一种理想的黏膜疫苗形式。
2.5 易于多价疫苗的构建 Audouy等基于BLPs展示平台分别构建了表面展示肺炎链球菌抗原蛋白IgA1p、PpmA和SlrA的BLPs疫苗，通过将三种颗粒按一定比例混合，可制备成二价、三价肺炎链球菌疫苗[21]。由于来自乳酸菌自溶素AcmA的PA蛋白与肽聚糖的非共价结合无特异性，两种或两种以上融合蛋白均可同时锚定于BLPs表面，制备成多价疫苗。随后该团队将肺炎球菌的PpmA、SlrA和IgA1p与PA融合蛋白表达后，任意二者或者三者按一定比例与裸露BLPs孵育后均可锚定在BLPs表面，制备为表面同时展示肺炎链球菌抗原蛋白的二价或三价BLPs疫苗[14]。
3 BLPs表面展示系统的应用 3.1 作为疫苗佐剂 季节流感病毒A/Wisconsin (H3N2)的HA亚单位疫苗添加GEM颗粒，鼻内免疫小鼠后诱导产生HI抗体的效价 > 40，相当于肌肉注射免疫的水平，比无佐剂组诱导产生的抗体水平更高、持续期更长，而且可诱导局部SIgA的产生[22]。另外，0.04 μg流感病毒A/PR/8/34 (H1N1)的HA亚单位疫苗添加GEM颗粒可诱导产生与1-5 μg无佐剂HA亚单位疫苗相当的HI抗体水平[23]。商品化的甲型H1N1流感病毒裂解疫苗添加GEM佐剂鼻内免疫小鼠可显著提高血清特异性IgG和HI抗体水平，无论在近处的肺、鼻腔黏膜还是较远的阴道黏膜位点均可诱导产生SIgA，保护小鼠抵御同源和异源强毒株的致死性攻击，且攻毒后肺脏病毒载量较无佐剂组降低100倍[24]。目前，乳酸菌GEM颗粒作为季节性流感亚单位疫苗佐剂，取得了良好的应用效果，已完成临床1期评价[4]。
3.2 作为黏膜疫苗递送载体 黏膜是机体的第一道防线，有效的黏膜免疫能够在病原入侵开始就起到防护作用，阻止病原进一步感染。黏膜疫苗接种无需带针注射，免疫副反应少，且适合大群免疫、多次免疫，是预防疾病感染的最佳途径。BLPs因其安全、高效的独特优势，多种呼吸道、消化道传染病BLPs黏膜疫苗已成功制备，均显示出良好的免疫保护效果(表 1)。表面展示呼吸道合胞体病毒(RSV) F蛋白的BLP-RSVF通过鼻腔黏膜免疫Balb/c小鼠或棉鼠，能诱导产生比灭活苗更高水平的鼻腔SIgA和血清中和抗体，受强毒攻击后能显著减少肺脏病毒载量[25]，成为预防RSV安全而有效的候选疫苗，目前已获得英国药物临床试验许可。
表 1. BLPs黏膜疫苗免疫原性与免疫保护力 Table 1. Immunogenicity and immunological protection of mucosa vaccine based BLPs
| Pathogen | Displayed antigens | Vaccination route | Animal model | Tested parameter | Observed outcome | |
| Virus | Influenza virus | HA | i.n. | Mouse | Immune responses and correlate of protection | Serum HI titers > 40, strong increase compared to i.m. commodity vaccine[4] |
| HA1 | i.g. | Mouse | Immune responses and lethal challenge | Significant level of serum IgG and sIgA. Survival rate of mice was up to 88% (7/8)[26] | ||
| M2e | i.n | Mouse | Immune responses and i.n. challenge | 100% protection, strong reduction of lung viral load[4] | ||
| NP | i.n | Mouse | Cellular response | Th1/Th2 balanced cellular response (IFN-γ/IL4 ratio)[4] | ||
| RSV | F | i.n | Mouse, cottonrat | Local mucosal response and correlate of protection | sIgA in the nose and neutralizing antibodies in sera. Strong reduction in lung virus titers upon RSV challenge[25] | |
| Bacteria | Streptococcus pneumoniae | IgA1p, SlrA or/and PpmA | i.n., i.m. | Mouse | Immune responses and Pulmonary and i.n. challenge | 50%-75% protection associated with strong reduction in bacteremia. Strong reduction in nasopharyngeal colonization[14] |
| PspA | i.n. | Mouse | Immune responses and i.n. challenge | PspA-specific IgG in sera and sIgA in mucosal washes. 100% protection against homologous and heterologous pneumococcal strain[27] | ||
| Plym2 | i.n. | Mouse | Both systemic and mucosal immune responses | High level of serum IgG antibodies and sIgA antibodies in lung lavages[18] | ||
| Yersinia pestis | LcrV | i.n./i.g. | Mouse | Immune responses and i.v. Challenge | 100% protection and 85% protection respectively[15] | |
| Shigella spp. | IpaB or/and IpaD | i.n. | Mouse | Immune responses and Pulmonary challenge | 100% protection against S. flexneri in adults and partial protection in newborns; 90% cross-protection against S. sonnei[17] | |
| Campylobacter jejuni | CjaA or/and CjaD | i.n./s.c./in ovo | Chickens chicken eggs | Immune responses and oral challenge | No protected effect i.n. or s.c. inoculation against intestinal tract colonization of wild type C. jejuni strain, while administered in ovo the mean level was reduced 100 times and correlate of significant levels of protection[28] | |
| Helicobacter pylori | CUE | i.n. | Mouse | Immune responses and i.n. challenge | Urease-specific antibody and local Th1/Th17 cell-medicated immune response. Strong reduction in the urease activity, gastric inflammation level and bacterial colonization[29-30] | |
| Parasites | Plasmodium berghei | CSP | i.m. | Mouse | Immune responses and Infected mosquito challenge | 100% protection[31] |
| i.m.: intramuscular; i.n.: intranasal; i.g.: intragastric; i.v.: intravenous; s.c.: subcutaneous. | ||||||
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3.3 作为病毒抗原纯化方法 通常病毒纯化的方法主要有：超速离心沉淀法(密度梯度)、超滤和分子排阻色谱(分子大小)、离子交换色谱(电荷)及亲和层析(特异性结合)，这些方法费时费力，且无法保证纯化的纯度与得率，将其应用于疫苗生产存在许多局限性，尤其培养液中杂蛋白、核酸、脂类、糖类等异源物质易引起免疫抑制、免疫副反应等。国内侯继波研究员团队通过将能与病毒特异性结合的接头分子融合PA结合于BLPs表面，与病毒的细胞培养物共孵育后，借助接头蛋白将病毒抓取下来，实现对病毒抗原的一步纯化。如将O型口蹄疫病毒(FMDV)的纳米抗体与PA序列融合经大肠杆菌串联表达后，锚定结合于BLPs表面，取适量与病毒培养物共孵育，简单的一步低速离心后收获的沉淀即为纯化后的FMDV抗原，其回收效率可达到99%，杂蛋白去除率达到90%[32]，动物免疫试验结果显示纯化后的FMDV具有良好的免疫原性；通过将与病毒糖蛋白特异性结合的胶原样凝集素与PA融合表达后锚定于BLPs表面，实现对猪繁殖与呼吸综合征病毒(PRRSV)的纯化，并且该病毒纯化体系具有广泛的适用性，可以用来纯化所有与凝集素结合的病毒[33]。
3.4 用于固定化酶与生物转化 多年来，表面展示的生物技术的另一个重要应用就是构建全细胞生物催化剂。纯化的游离或固定化酶用于传统酶促反应，由于酶生产与纯化成本较高，固定化是有效的策略。传统的做法是将酶展示在经遗传修饰的产生细胞表面，但是由于暴露不完全或错误折叠可能会降低酶的活性。为了解决这一问题，将活性酶融合锚定序列经适宜的宿主表达后形成正确的构象，再结合于乳酸菌BLPs上，这样酶会以高活性的形式完全展示在肽聚糖骨架表面。枯草杆菌蛋白酶QK-2能降解纤维蛋白，是一种有效的血栓溶解剂，通常纯化于枯草芽孢杆菌QK02培养上清，但复杂的纯化方法限制了其广泛应用，Mao等通过将其C端融合PA序列分泌表达后，结合于BLPs表面，所形成的展示颗粒依然保持其纤维蛋白溶解活性，有望广泛应用于血栓形成性疾病的防治中[34]。同样，Bosma等将地衣杆菌α-淀粉酶与PA融合表达后可结合并展示于BLPs表面，并且融合有PA的地衣杆菌α-淀粉酶和大肠杆菌β内酰胺酶可同时展示在BLPs表面，并保持各自的酶活[3]。
4 展望 细菌样颗粒作为一种新型乳酸菌表面展示技术，近年来的快速发展和在疫苗研制、佐剂开发、蛋白纯化等方面的广泛应用已充分表明该技术具有巨大的发展潜力，尤其在黏膜疫苗的研发上可望与病毒样颗粒疫苗一道成为最有前景的基因工程亚单位疫苗形式。但目前BLPs仍属新兴领域，有关家禽BLPs黏膜疫苗的有效性及其相关免疫机制的报道尚不多见。尽管现已证实BLPs可被哺乳动物的TLR2分子所识别，但家禽的免疫系统在某些方面与哺乳动物不同，家禽的chTLR2具有chTLR2t1 (chTLR2 type 1)和chTLR2t2 (chTLR2 type 1)两种亚型，二者可分别与chTLR1LA (chTLR1-like protein A)和chTLR1LB (chTLR1-like protein B)形成4种类似哺乳动物TLR2/TLR1或TLR2/TLR6的异源二聚体，因此，BLPs作为家禽黏膜疫苗的研发形式其有效性如何、通过何种分子对其进行免疫识别成为当前亟需解决的科学问题。作者所在实验室目前正在以家禽新城疫病毒为模型，构建BLPs黏膜疫苗，以期展现BLPs在家禽上的广阔应用前景，为家禽传染病黏膜疫苗和黏膜佐剂的研发提供有力借鉴。在外源融合蛋白的制备上，较为常见的表达宿主有大肠杆菌、乳酸菌、昆虫细胞、哺乳动物细胞等，但目前所制备的BLPs疫苗其抗原蛋白多以非糖基化蛋白为主，广泛表达于大肠杆菌、乳酸菌等原核宿主中，但对于某些病原其保护抗原蛋白为表面糖蛋白(如流感病毒的HA蛋白)，实现对糖蛋白的高效可溶性表达和最大程度地保持免疫原的天然构象，也是今后BLPs疫苗研发的重要突破方向。随着BLPs理论研究的不断深入，以及未来蛋白表达与展示相关技术和工艺的不断成熟，基于BLPs的微生物表面展示技术必将在更多领域具有广阔的应用前景。
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