药学学报

Pathogen	Polysaccharide	Non-sugar moiety	Phase of clinical trial	Company	Technical type	Ref.
Shigella	O-polysaccharides of S. flexneri 2a	rEPA	Phase Ⅱb NCT02646371	Limmatech Biologics	PGCT	[8]
O-polysaccharide of S. dysenteriae 1	rEPA	Phase Ⅰ NCT01069471	Limmatech Biologics	PGCT	[9]
O-polysaccharide of S. sonnei	OMV	Phase Ⅱb NCT03527173	GSK	GMMA	[10]
O-polysaccharides of S. flexneri 2a, 3a, and 6, and S. sonnei	rEPA	Phase Ⅱb NCT06615375	Limmatech Biologics	PGCT	[11]
O-polysaccharides of S. flexneri 2a	TT	Phase Ⅱ NCT04602975	Institute Pasteur	Semi-synthetic glycoconjugate vaccines	[12]
O-polysaccharides of S. flexneri 1b, 2a, and 3a and S. sonnei	OMV	Phase Ⅱ NCT06663436	GSK	GMMA	[13]
Streptococcus pneumoniae	Capsular polysaccharide of serotype 12F	rEPA	Phase Ⅰ NCT03303976	Limmatech Biologics	PGCT	[14]
Capsular polysaccharides of 24 serotypes	CP1 fusion protein	Phase Ⅱ NCT03803202	Affinivax	Multiple antigen presenting system	[15]
Capsular polysaccharides of 24 serotypes	eCRM197	Phase Ⅱ NCT05297578	Vaxcyte	Click chemistry-based conjugation via non- natural amino acids	[16]
Salmonella	O-polysaccharides of S. typhimurium and Salmonella enteritidis	OMV	Phase Ⅰ/Ⅱa NCT05480800	GSK	GMMA	[17]
Vi-capsular polysaccharide of S. Typhi	CRM197	Phase Ⅰ NCT03926455	Matrivax	Protein capsular matrix vaccine	[18]
Extraintestinal pathogenic Escherichia coli	O-polysaccharides of serotypes 1A, 2, 6A, and 25B	rEPA	Phase Ⅱ NCT02546960	Janssen Research & Development, LLC	PGCT	[19]
O-polysaccharides of 9 serotypes	rEPA	Phase Ⅲ NCT04899336	Janssen Research & Development, LLC	PGCT	[20]
O-polysaccharides of 10 serotypes	rEPA	Phase Ⅰ/Ⅰia NCT03819049	Janssen Research & Development, LLC	PGCT	[21]

Pathogen	Polysaccharide	Non-sugar moiety	Phase of clinical trial	Company	Technical type	Ref.
Shigella	O-polysaccharides of S. flexneri 2a	rEPA	Phase Ⅱb NCT02646371	Limmatech Biologics	PGCT	[8]
O-polysaccharide of S. dysenteriae 1	rEPA	Phase Ⅰ NCT01069471	Limmatech Biologics	PGCT	[9]
O-polysaccharide of S. sonnei	OMV	Phase Ⅱb NCT03527173	GSK	GMMA	[10]
O-polysaccharides of S. flexneri 2a, 3a, and 6, and S. sonnei	rEPA	Phase Ⅱb NCT06615375	Limmatech Biologics	PGCT	[11]
O-polysaccharides of S. flexneri 2a	TT	Phase Ⅱ NCT04602975	Institute Pasteur	Semi-synthetic glycoconjugate vaccines	[12]
O-polysaccharides of S. flexneri 1b, 2a, and 3a and S. sonnei	OMV	Phase Ⅱ NCT06663436	GSK	GMMA	[13]
Streptococcus pneumoniae	Capsular polysaccharide of serotype 12F	rEPA	Phase Ⅰ NCT03303976	Limmatech Biologics	PGCT	[14]
Capsular polysaccharides of 24 serotypes	CP1 fusion protein	Phase Ⅱ NCT03803202	Affinivax	Multiple antigen presenting system	[15]
Capsular polysaccharides of 24 serotypes	eCRM197	Phase Ⅱ NCT05297578	Vaxcyte	Click chemistry-based conjugation via non- natural amino acids	[16]
Salmonella	O-polysaccharides of S. typhimurium and Salmonella enteritidis	OMV	Phase Ⅰ/Ⅱa NCT05480800	GSK	GMMA	[17]
Vi-capsular polysaccharide of S. Typhi	CRM197	Phase Ⅰ NCT03926455	Matrivax	Protein capsular matrix vaccine	[18]
Extraintestinal pathogenic Escherichia coli	O-polysaccharides of serotypes 1A, 2, 6A, and 25B	rEPA	Phase Ⅱ NCT02546960	Janssen Research & Development, LLC	PGCT	[19]
O-polysaccharides of 9 serotypes	rEPA	Phase Ⅲ NCT04899336	Janssen Research & Development, LLC	PGCT	[20]
O-polysaccharides of 10 serotypes	rEPA	Phase Ⅰ/Ⅰia NCT03819049	Janssen Research & Development, LLC	PGCT	[21]

创新技术在多糖结合疫苗研发中的应用进展

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李湘 , 刘睿昊 , 黄文华 , 王恒樑 ^* , 朱力 ^*

药学学报 | 专题报道: 糖疫苗的研究进展与发展趋势 2025,60(7): 1991-2001

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药学学报 |专题报道: 糖疫苗的研究进展与发展趋势 2025 , 60 (7) : 1991 -2001

创新技术在多糖结合疫苗研发中的应用进展

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李湘, 刘睿昊, 黄文华, 王恒樑^*, 朱力^*

作者信息

军事科学院军事医学研究院, 病原微生物生物安全全国重点实验室, 北京 100071

通讯作者:

王恒樑, E-mail: wanghl@bmi.ac.cn

朱力, E-mail: jewly54@bmi.ac.cn

作者简介:

作者贡献:

李湘和刘睿昊完成文献检索和文章撰写; 黄文华对文章内容进行客观核对; 王恒樑和朱力对文章进行指导和修改。

Progress in the development of conjugate vaccines using innovative technologies

Xiang LI, Rui-hao LIU, Wen-hua HUANG, Heng-liang WANG^*, Li ZHU^*

Affiliations

State Key Laboratory of Pathogen and Biosecurity, Academy of Military Medical Sciences, Academy of Military Sciences, Beijing 100071, China

出版时间: 2025-07-12 doi: 10.16438/j.0513-4870.2025-0353

文章导航

摘要

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多糖结合疫苗是预防细菌性传染病的重要手段, 目前已上市的多糖结合疫苗均采用分步提取天然多糖抗原、纯化载体蛋白后, 体外化学交联方法生产。近年来, 合成生物学等新兴技术的快速发展, 催生了一系列创新型多糖结合疫苗生产设计策略。本文重点对蛋白多糖偶联技术和无细胞糖蛋白合成技术研究进展进行综述, 同时也简要介绍了膜抗原通用模块技术、半合成糖疫苗制备技术、非共价结合技术和基于非天然氨基酸的点击化学偶联技术在多糖结合疫苗研发中的应用。未来, 随着人工智能技术的发展, 有望在寡糖转移酶和载体蛋白的人工设计改造方面取得突破, 进一步提升多糖结合疫苗的免疫效果和生产效率。

关键词

多糖结合疫苗 / 合成生物学 / 蛋白多糖偶联技术 / 无细胞糖蛋白合成技术 / 膜抗原通用模块技术

Abstract

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Glycoconjugate vaccines are essential tools for preventing bacterial infectious diseases. Currently licensed glycoconjugate vaccines are produced through a stepwise process that involves the extraction of native polysaccharide antigens, purification of carrier proteins, and subsequent in vitro chemical conjugation. In recent years, the rapid advancement of emerging technologies such as synthetic biology has driven the development of innovative strategies for designing and manufacturing glycoconjugate vaccines. This review highlights progress in two representative technologies: protein glycan coupling technology (PGCT) and cell-free glycoprotein synthesis (CFGpS). It also briefly explores additional emerging approaches in glycoconjugate vaccine development, including generalized modules for membrane antigens (GMMA), semi-synthetic glycoconjugate vaccines, non-covalent coupling strategies, and click chemistry-based conjugation using non-natural amino acids. Looking ahead, advancements in artificial intelligence (AI) are expected to accelerate the computational design and engineering of oligosac-charyltransferases and carrier proteins, ultimately improving the immunogenicity and manufacturing efficiency of next-generation glycoconjugate vaccines.

Key words

glycoconjugate vaccine / synthetic biology / protein glycan coupling technology / cell-free glycoprotein synthesis / generalized modules for membrane antigen

引用本文

李湘, 刘睿昊, 黄文华, 王恒樑, 朱力. 创新技术在多糖结合疫苗研发中的应用进展. 药学学报, 2025 , 60 (7) : 1991 -2001 . DOI: 10.16438/j.0513-4870.2025-0353

Xiang LI, Rui-hao LIU, Wen-hua HUANG, Heng-liang WANG, Li ZHU. Progress in the development of conjugate vaccines using innovative technologies[J]. Acta Pharmaceutica Sinica, 2025 , 60 (7) : 1991 -2001 . DOI: 10.16438/j.0513-4870.2025-0353

正文

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多糖类物质是病原细菌重要的胞外聚合物, 其中脂多糖 (lipopolysaccharide, LPS) 和荚膜多糖 (capsular polysaccharide, CPS) 具有特异性免疫原性, 与细菌的致病性密切相关, 如肺炎链球菌 (Streptococcus pneumoniae)、脑膜炎奈瑟菌 (Neisseria meningitidis) 和肺炎克雷伯杆菌 (Klebsiella pneumoniae) 等。这些多糖不仅是细菌的重要毒力因子, 也是被广泛用于细菌疫苗研发的一类有效抗原^[1,2]。单独的多糖是T细胞非依赖性抗原, 其主要通过与B细胞表面的抗原识别受体结合, 激活B细胞并分化为可分泌抗体的浆细胞。这个过程并没有T细胞的参与, 因此不会形成免疫记忆, 产生的抗体也主要是亲和力较低的IgM和IgG2^[3,4]。另外, 多糖疫苗在免疫系统发育不完全的婴幼儿、免疫力低下的老年人群中无法产生有效的免疫保护^[4,5]。

多糖结合疫苗的出现为该问题提供了解决方案。1929年, Avery和Goebel证明与载体蛋白偶联可增强多糖的免疫原性, 这一发现为多糖结合疫苗的研发提供了理论基础^[6]。多糖结合疫苗的开发策略, 是通过将多糖与非糖单元 [蛋白质或外膜囊泡 (outer membrane vesicles, OMV) 等载体] 共价连接, 将多糖抗原转变为T细胞依赖性抗原, 诱导产生针对多糖抗原的高亲和力抗体并产生免疫记忆, 从而使婴幼儿产生有效的免疫应答。近几十年, 多糖结合疫苗显著降低了各种高致病性细菌引起的传染病的发病率, 也在很大程度上遏制了各种耐药菌的传播, 为全球公共卫生事业作出了重要贡献。

目前, 已经上市的多糖结合疫苗主要针对流感嗜血杆菌、肺炎链球菌及脑膜炎奈瑟球菌, 主要采用传统化学交联方法生产, 这种方法需要分别将病原细菌表面多糖抗原和特定载体蛋白进行分离和纯化。尽管这种方法已经成功用于多种疫苗的开发, 但由于其复杂的多步骤纯化流程, 造成疫苗的生产成本较高。例如, 13价肺炎链球菌疫苗Prevnar 13的生产涉及数百个步骤, 导致其市场价格对中低收入国家来说过于昂贵, 限制了这些疫苗的普及。在过去10年中, 蛋白多糖偶联技术 (protein glycan coupling technology, PGCT) 因其能够简化偶联物的生产过程, 成为多糖结合疫苗生产中一种极具吸引力的策略。PGCT的核心优势在于, 它可以在工程菌株体内一步完成生物偶联反应, 无需单独表达和纯化载体蛋白和多糖抗原。这种技术显著降低了疫苗生产的复杂性和成本, 为中低收入国家提供了更可及的疫苗选择。在此理论和成功实践的基础上, 结合无细胞蛋白合成(cell-free protein synthesis, CFPS) 技术, Jewett团队^[7]发展了无细胞糖蛋白合成 (cell-free glycoprotein synthesis, CFGpS) 技术, 使得蛋白质在体外合成的同时能够实现糖基化修饰, 进一步拓展了多糖结合疫苗的制备方法和应用范围。CFGpS技术不仅显著简化了疫苗生产工艺, 提高了生产速度和可控性, 还为全球疫苗制造和应急响应提供了一种高效、便捷的解决方案。

此外, 随着膜抗原通用模块 (generalized modules for membrane antigens, GMMA) 技术、半合成糖疫苗制备技术、非共价结合技术和基于非天然氨基酸的点击化学偶联技术等新兴平台的出现, 为多糖结合疫苗的生产提供了更多具有潜力的制备策略 (图1)。这些创新技术的应用, 将进一步推动该领域的发展与进步。总之, 过去10年, 各类新兴技术已应用于肺炎链球菌、志贺菌等候选多糖结合疫苗的生产, 部分产品已顺利进入临床试验阶段 (表1^[8-21])。

1　蛋白多糖偶联技术

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糖基化是蛋白质重要的翻译后修饰, 细菌中主要有蛋白N-糖基化和蛋白O-糖基化两种形式。1999年, 研究者首先在空肠弯曲杆菌中发现了蛋白质N-糖基化现象^[22]。2000年, 催化该过程的寡糖转移酶 (oligosaccharyl transferase, OSTase) PglB基因序列被明确^[23]。2005年, Feldman团队^[24]将这一蛋白质N-糖基化系统克隆转移到大肠杆菌中, 首次实现了大肠杆菌O7多糖抗原与载体蛋白ArcA的连接, 成功制备出第一个生物合成的多糖结合疫苗候选物, 为低成本、高效的多糖-蛋白偶联物生产提供了经典范例。这种基于寡糖转移酶系统的技术被称为PGCT。该技术通过将病原菌的多糖合成基因簇、载体蛋白和OSTase在工程菌株中共表达, 由OSTase催化多糖与载体蛋白结合, 一步法生成多糖-蛋白偶联物 (图2), 显著地简化了疫苗生产步骤, 提高了生产安全性。关于该技术体系已经有较为详细的综述报道^[25], 本文仅对工程菌株的特点进行简要补充。

PGCT的经典策略是将目标多糖合成基因簇克隆至大肠杆菌中进行异源表达^[26-28]。为优化多糖结合疫苗的生产, 研究人员对大肠杆菌进行了系统性改造。首先, 通过系统性地敲除宿主菌自身多糖合成相关基因来提升异源多糖的合成效率。2005年, Feldman团队^[24]敲除了W3110菌株的O-抗原连接酶基因waaL, 该基因负责将O-多糖转移至脂质A核心, 其缺失显著提高了OSTase的催化效率。在此基础上, 作者团队又敲除了W3110 △waaL菌株自身的OPS合成相关基因 (从wbbH到wbbL的5个基因), 进一步提高了蛋白质糖基化效率^[29]。2005年, Linton团队^[30]敲除了W3110菌株的wecA基因, 该基因催化GlcNAc转移至十一异戊烯醇焦磷酸 (undecaprenyl pyrophosphate, Und-PP) 上, 启动肠杆菌共同抗原 (enterobacterial common antigen, ECA) 及多种大肠杆菌O抗原的合成, 因此, 敲除wecA能够显著提升还原端非GlcNAc的病原细菌多糖的异源表达。2024年, Jorgenson团队^[31]通过敲除MG1655菌株中编码磷酸糖基转移酶及引发聚糖组装的糖基转移酶的基因 (wecA、wcaJ、gtrB和arnC 4个基因), 并过表达Und-PP合成酶UppS, 增加了可用的Und-PP, 显著提高了异源多糖的表达量。在遗传稳定性改造方面, 有报道将PGCT中的关键元件 (如OSTase和多糖合成基因簇) 整合至大肠杆菌的染色体上, 这样既能够减轻了宿主代谢负担, 又提高了元件的稳定性, 从而促进糖蛋白生产^[32-34]。在分泌表达改造方面, 研究人员通过敲除Braun脂蛋白基因, 构建了一种“渗漏型”大肠杆菌菌株, 其能够将糖蛋白分泌至培养基中, 不仅提高了产量, 还简化了纯化流程^[35]。

尽管大肠杆菌已广泛用于生产针对多种病原细菌的多糖结合疫苗, 但是病原细菌的多糖合成基因簇克隆 (通常 > 10 kb) 和高效异源表达通常需要进行繁杂的优化过程。因此, 直接利用减毒病原微生物作为宿主菌的潜力也逐渐显现。首先, 借助细菌抗原多糖的结构相似性, 使用低毒性工程菌发酵生产高等级生物安全风险细菌疫苗已被证明是一种很好的研发策略。例如, 小肠结肠炎耶尔森菌O:9的O抗原结构与流产布鲁氏菌O抗原高度相似, 可以被用做生产抗布鲁菌疫苗的工程菌株, 能够规避直接使用布鲁菌发酵所带来的安全风险^[36-38]。此外, 为了简化多糖结合疫苗的生产, 研究者开发了一种新策略: 利用病原细菌内生的高效多糖合成过程, 通过基因改造 (敲除O抗原连接酶waaL基因) 构建LPS缺陷的减毒病原菌, 作为生物反应器来合成目标糖蛋白^[39,40]。例如, 肺炎克雷伯菌菌株ATCC 25955因生长速率快, 具有被改造为肺炎克雷伯菌糖疫苗工程底盘菌株的潜力。作者团队通过敲除该菌的荚膜多糖合成基因簇和O抗原连接酶基因waaL, 构建了两种不同血清型的糖基工程菌株^[41]。但是该策略需要对不同病原细菌进行减毒, 并探索不同细菌的大规模培养条件。因此, 该策略更适用于肠杆菌科细菌疫苗的开发, 因为这类细菌具有相对成熟的基因编辑体系, 并且其代谢系统与大肠杆菌极其相似, 发酵条件优化工作难度不大。相比之下, 对于土拉弗朗西斯菌、类鼻疽伯克霍尔德菌等培养条件苛刻、致病性强的特殊病原体而言, 该策略在其疫苗研发中的应用价值则会较为有限。

2　无细胞糖蛋白合成技术

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CFPS是一种在体外环境下实现蛋白质转录与翻译的技术, 近年来已成为加速蛋白设计、生物制造与功能测试的重要平台。CFPS系统依赖细胞裂解物中的转录与翻译机制, 无需完整细胞即可高效合成目标蛋白。与传统的细胞培养系统相比, CFPS具有更短的生产周期、更高的可调控性, 并能够表达难以在细胞内表达的蛋白, 因此在疫苗研发、蛋白工程和合成生物学领域展现出巨大的应用潜力。作为CFPS技术的延伸, CFGpS进一步拓展了其应用范围, 使得蛋白质在体外合成的同时能够实现糖基化修饰。

事实上, CFGpS是前述PGCT技术与CFPS技术的联合运用。CFGpS同样是利用基因工程改造的大肠杆菌等工程菌株 (如前文所述W3110 △waaL等), 使其在培养过程中合成脂质连接寡糖 (lipid-linked oligosaccharides, LLOs) 并表达OSTase。这些糖基化相关的酶和底物, 在细胞裂解物的制备过程中得以保留, 从而使目标蛋白被无细胞系统翻译后, 能够直接进行糖基化修饰 (图3)。这一技术避免了传统PGCT受宿主细胞不同空间限制的问题 (如目标蛋白需要定位至细菌周间质), 从而实现了蛋白糖基化的灵活可控。

与传统的多糖结合疫苗生产方法相比, CFGpS技术具有显著优势: ① 生产灵活, 快速响应: CFGpS系统可在短时间内高效合成目标蛋白, 并适应不同疫苗抗原的需求, 特别适用于应急疫苗生产; ② 高效可控的糖基化修饰: 通过优化无细胞裂解液, 可精确调控OSTase和糖供体的种类, 实现特定糖基化模式的定向修饰; ③ 冻干存储, 便携式制造: 无细胞系统可以冻干储存, 在需要时仅需加水激活即可完成蛋白合成和糖基化修饰, 大大提高了疫苗生产的可及性, 特别适用于资源受限地区的疫苗分发。

该技术的应用使得不同病原体的多糖抗原能够在体外进行精准的糖基化修饰, 最终获得具有优良免疫原性的糖蛋白。本文将较为详细地综述CFPS技术在多糖结合疫苗中的应用, 重点分析其在疫苗生产中的优势与挑战。

2.1　CFGpS技术的创立

2018年, 美国西北大学Jewett团队^[7]开发了一种创新的一锅法CFGpS系统, 该系统能够在同一反应体系中同时完成蛋白合成与N-糖基化修饰, 从而提高糖基化蛋白的合成效率和一致性。传统的糖蛋白合成, 需要在蛋白合成后将蛋白分离, 再进行糖基化修饰; 而CFGpS系统与之相比的最大优势, 就是利用改造过的大肠杆菌CLM24菌株制备细胞裂解液, 直接在细胞裂解液中整合了OSTase和LLOs, 通过OSTase识别目标蛋白上的特定糖基化位点, 实现了蛋白合成与糖基化修饰的同步高效启动。这一改进使得无细胞体系能够用于结合疫苗的高效生产, 展现出广泛的应用潜力。

Jewett团队^[24]首先探讨了CFPS系统的反应条件, 选用了基因工程改造的大肠杆菌CLM24菌株, 该菌株通过敲除waaL基因, 确保了在无细胞系统中糖基化修饰能够高效、特异性地发生在目标蛋白上; 随后, 该团队还优化了OSTase和LLOs的富集实验方案, 验证了在不补充额外的OSTase和LLOs的情况下, 通过单纯使用富集的细胞裂解物即可实现载体蛋白的糖基化, 无需额外的复杂操作步骤; 最后, 该团队评估了CFGpS系统在不同蛋白底物上的适用性, 发现该系统能够糖基化多种蛋白, 包括抗体片段、绿色荧光蛋白和人促红细胞生成素等。表明该系统能够适应不同类型的蛋白, 且在糖基化修饰的均一性 (糖基化效率接近100%) 和稳定性方面优于传统的活细胞系统。

CFPS作为一种快速反应、模块化的技术, 通过将反应体系中的组分进行冻干处理, 能够实现在室温下进行运输。到达目的地后, 只需简单加水激活反应系统, 即可在48 h内完成载体蛋白的合成和蛋白的糖基化, 迅速制备出多糖结合疫苗。这一方法不仅大大缩短了疫苗的制备时间, 同时也解决了冷链运输的问题, 使得多糖结合疫苗的生产、部署和运输更加灵活和高效。

2.2　CFGpS技术的优化发展

2021年, Jewett团队^[42]通过改进工程细菌裂解液的制备方法, 进一步富集膜囊泡和膜结合糖基化酶 (如寡糖转移酶PglB和PglO), 显著提高了糖基化蛋白的合成效率和一致性。与传统无细胞系统中细胞裂解物的制备方法相比, 采用均质破碎方式对细胞进行破碎, 能够降低对高转速离心的依赖, 并且有效提高了膜囊泡浓度。这一改进不仅提高了膜囊泡的质量, 还降低了对细胞裂解物的制备要求, 使更多研究团队能够采用无细胞系统进行多糖结合疫苗的生产。研究进一步优化了细胞裂解物的处理方法, 发现通过调节离心速度和提取条件, 尤其是在低速离心 (S12提取法) 中, 膜囊泡浓度得到了显著提升。与传统的高速离心 (S30提取法) 相比, S12提取物中的膜囊泡浓度提高了1.2～2倍。这一结果表明, 低速离心与均质破碎联用方法, 能够有效避免膜囊泡的过度破裂或损失, 从而保留了更多完整的膜结构, 并确保膜结构正确取向 (即保留了更多用于蛋白糖基化的活性成分), 显著提高了蛋白质糖基化效率。无论是在O-连接还是N-连接糖基化糖蛋白的合成过程中, 优化后的S12提取物相比传统的S30提取物, 都能够显著提高糖蛋白的产量和糖基化效率。此外, 该研究还进一步探讨了CFGpS系统的可调控性, 证明了通过调节膜囊泡中的OSTase和LLOs的种类, 可以灵活调整糖基化模式, 以满足不同糖基化需求。这一高度的可调控性使得CFGpS系统在疫苗生产中的应用更加灵活和多样化, 能够高效生产不同类型的糖基化疫苗。

2.3　CFGpS技术的剂型研究

无细胞糖蛋白合成系统的另一大优势就是具有独特的按需制造特点和极佳的冻干稳定性, 这使得它在结合疫苗的生产中展现出巨大的潜力。传统的疫苗生产通常依赖活细胞培养, 这不仅需要复杂的培养设施和长时间的生产周期, 还受限于冷链运输的要求, 限制了疫苗的快速分发和使用。通过优化的冻干技术, 无细胞系统能够在不依赖冷链运输的情况下, 将反应体系储存并稳定地运送到全球任何地点, 从而在资源有限的环境中实现快速疫苗生产。最近报道的基于CFGpS技术建立的结合疫苗体外表达平台, 能够在数小时内完成所需疫苗的合成^[43], 并且能够根据实际需求迅速调整反应规模, 极大地缩短了生产周期。例如, 在面对突发的传染病时, 疫苗可以通过简易的复溶操作迅速启动生产, 无需提前储备大量的疫苗库存^[44]。

冻干稳定性是上述平台在疫苗生产中的一个关键优势。研究表明, 加入麦芽糊精 (maltodextrin) 作为冻干保护剂和能量来源, 能够显著提高无细胞系统的稳定性和反应效率。麦芽糊精不仅能在冻干过程中保护无细胞反应体系免受热和冰冻损伤, 还能作为低成本的能量来源, 替代传统的磷酸化二级能量底物 (如磷酸烯醇丙酮酸)。实验结果证明, 使用麦芽糊精加入无细胞反应体系冻干后, 在不同温度条件下 (包括高达50 ℃的环境) 存储4周后, 能有效维持无细胞反应体系的活性和稳定性。简言之, 结合疫苗体外表达平台将CFGpS技术的按需制造特点与冻干稳定性相结合, 不仅能够实现低成本、高效的生产, 而且能够在高温条件下存储和运输, 为疫苗生产提供了一个革命性的解决方案, 对于疫苗的分发和储存至关重要, 尤其是在资源匮乏的地区或突发公共卫生事件中。

2.4　CFGpS技术的发展方向

随着技术进步, 大规模生产的实现将成为无细胞糖基化技术的下一步目标。尽管当前无细胞系统在小规模生产中表现优异, 但在大规模应用中仍面临一些挑战, 如生产规模的扩展、反应效率的优化、原料成本的降低等。预计随着工程细菌裂解液提取物的制备、糖基化反应的催化条件优化及系统稳定性的增强, 无细胞糖基化技术将在未来实现更高效、更经济的大规模生产, 尤其在应急疫苗和特种疫苗的生产中, 发挥更大作用。展望未来, 无细胞糖基化系统的应用还将扩展到更广泛的生物制剂生产领域, 不仅限于多糖结合疫苗, 还包括抗体工程、癌症疫苗、抗病毒疫苗等领域。

3　膜抗原通用模块技术

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除了传统以蛋白质为载体, 通过直接交联载体蛋白和多糖抗原外, 基于脂膜类载体递送多糖抗原也是一种很有前途的策略。细菌的OMV是一种具有天然佐剂效应的纳米颗粒: 其携带的LPS、肽聚糖和鞭毛蛋白等病原体相关模式分子可激活免疫反应^[45], 同时其纳米尺度的粒径也易于被抗原呈递细胞摄取, 促进抗原递送, 逐渐成为了新型多糖疫苗开发的理想载体^[46,47]。然而, 细菌天然释放的OMV通常具有较高的毒性 (来自内毒素LPS), 且产量较低, 难以满足疫苗生产需求。有研究人员通过去污剂提取法提高了OMV的产量, 并降低了LPS含量以实现减毒^[48-50], 但是去污剂可能破坏重要外膜蛋白 (例如PorA等关键抗原) 的天然构象, 导致免疫原性降低^[51,52]。为此, 研究者开发了GMMA技术, 即通过基因编辑方法, 改造OMV特性: 敲除抑制OMV分泌的相关基因 (如TolR) 以提高产量^[53], 同时修饰脂质A结构 (减少酰基链和磷酸基团) 以降低炎症反应^[45,54-57]。目前, 基于上述技术, 已经陆续研发了针对志贺菌、沙门菌和链球菌等病原体的候选疫苗^[17,58,59]。

除了GMMA技术, 研究者还利用大肠杆菌OMV表达异源多糖, 建立了糖工程OMV (glyOMV) 平台, 进一步拓展了OMV作为疫苗平台的潜力^[60]。具体来说, 是将目的多糖合成基因簇导入大肠杆菌, 合成的异源多糖被组装到LPS中并随其掺入OMV中^[48]。这种策略使OMV成为一个“即插即用”的平台, 可以展示来自不同病原菌的多糖抗原。使用这种方法, Chen等^[61]成功构建了一组glyOMV, 展示了来自8种病原菌菌株的O-多糖, 其中包括高毒力的土拉弗朗西斯菌亚种 (A型) 菌株Schu S4。实验结果显示, 利用glyOMV平台制备的针对Schu S4的多糖结合疫苗诱导了高滴度的抗体并具有良好的保护效果。此后, 研究人员利用该技术平台, 成功开发了针对多种病原菌的候选疫苗, 包括肺炎链球菌、金黄色葡萄球菌和空肠弯曲菌等^[62,63]。

作者团队综合了两种策略的优点开发了糖工程外膜囊泡 (glycoengineered GMMA, geGMMA), 即首先通过敲除大肠杆菌的lpxM基因来进行减毒, 同步敲除内源OPS合成相关基因 (wbbH-wbbL基因簇), 从而实现GMMA体系的“减毒增产”效果; 并利用改造后的大肠杆菌, 导入肺炎克雷伯菌O1和O2的O-抗原多糖生物合成基因簇, 实现了glyOMV平台的“异源表达”效果。实验结果显示, 利用geGMMA策略制备的新型糖疫苗能够激发高滴度的抗体反应, 具有良好的免疫保护效果。

4　半合成糖疫苗制备技术

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糖链化学合成技术的快速进步, 催生了半合成糖疫苗制备技术, 为多糖结合疫苗研发开辟了一条全新路径。半合成糖疫苗制备技术是指通过化学合成方法制备特定的糖抗原片段, 然后将其与载体蛋白质结合, 形成结合疫苗的一种新兴技术 (图4)。相比传统方法中从病原菌中直接提取多糖, 半合成糖疫苗中的糖抗原是完全通过化学合成获得的, 不仅避免了病原菌大规模发酵带来的生物安全风险, 并且能够通过精确调控多糖的结构和重复单位的长度来提高疫苗的均一性和有效性^[64-67]。该领域的首个里程碑事件是Quimi-Hib疫苗的成功研发, 其采用化学合成的8聚核糖基-核糖醇-磷酸 (polyribosyl-ribitol-phosphate, PRP) 片段与破伤风类毒素蛋白偶联, 开创了人工半合成糖疫苗的先河^[68]。2009年, 法国巴斯德研究所Mulard团队^[69]化学合成了福氏志贺菌2a的O-多糖 (包含3个重复单元), 与载体蛋白TT偶联后开发了一种针对福氏志贺菌2a的半合成糖疫苗。该疫苗在Ⅰ期临床试验中显示出良好的安全性和耐受性^[70]。2020年, 该疫苗进入Ⅱ期临床试验。2024年, 我国华东理工大学的研究团队^[71]也利用这种半合成糖疫苗技术, 开发了一种基于甘露糖醛酸四糖的铜绿假单胞菌糖疫苗, 能够引发强烈而特异性的免疫反应, 对不同类型的铜绿假单胞菌菌株具有广泛的保护作用。

在糖链人工合成领域, 传统合成聚糖的方法主要有化学法和酶法^[72], 而自动聚糖组装 (automated glycan assembly, AGA) 技术的出现大大加速了聚糖的合成, 成为聚糖合成的重要策略^[73-75]。2001年, Seeberger课题组^[76]开始研究基于固相聚糖的自动合成方法。2018年, Zhang等^[77]实现了基于酶法的聚糖自动合成。虽然两种方法都取得了一定进展, 但是两者都是通过单糖逐个累加的方式使糖链延伸 (加法式合成), 合成效率较低, 在合成尺寸较大的聚糖上也存在挑战。北京大学叶新山团队^[78]在液相聚糖自动合成领域的重大研究成果突破了这一技术瓶颈。2004年, 该团队提出基于“糖基供体预活化”的一釜寡糖合成新策略。2017年, 该团队基于该原理合成了由92个糖单元组成的阿拉伯半乳聚糖^[79]。在此基础之上, 2022年该团队又研制出国际首台双模式液相糖自动合成仪, 同时利用自动乘法合成策略成功合成了由1 080个单糖单元组成的阿拉伯聚糖分子^[80], 将合成的多糖尺寸提升到了一个新的高度, 也为合成更为复杂的病原细菌多糖抗原提供了颠覆性技术平台, 有望加速促进半合成糖疫苗的品种研发。

5　非共价结合技术

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在过去10年中, 就多糖和非糖单元的连接而言, 除了共价偶联方法外, 还出现了一些新的非共价偶联策略。其中一种策略是多抗原呈递系统 (multiple antigen presenting system, MAPS)。该平台利用生物素与根瘤菌亲和素之间的非共价相互作用, 将多糖和病原的蛋白抗原组装成大分子复合物 (图5)。这种复合物能够模拟全细胞疫苗的特性, 从而提供与全细胞疫苗相当的免疫效果^[81]。例如, 利用肺炎链球菌14型荚膜多糖和不同病原特异性蛋白抗原组装的MAPS复合物均能够激发强烈的免疫反应^[82]。基于MAPS技术平台, Affinivax公司开发的24价肺炎链球菌候选疫苗AFX3772在Ⅱ期临床试验中展现出与已上市的Prevnar 13相当的安全性和免疫原性^[15]。基于这一积极的Ⅱ期临床试验数据, 美国FDA已授予AFX3772突破性疗法认定。

另一种类似的策略是蛋白质荚膜基质疫苗 (protein capsular matrix vaccine, PCMV)。该技术通过将多糖抗原包埋在交联的蛋白质基质中, 形成稳定的疫苗结构^[83,84]。研究表明, 使用肺炎链球菌14型荚膜多糖和蛋白质基质制备的PCMV能够诱导高滴度的抗体反应, 显示出良好的免疫效果^[83]。Matrivax公司基于该技术平台开发的伤寒沙门菌候选疫苗Typhax, 在Ⅰ期临床试验中显示出了良好的安全性和免疫原性。结果显示, 在2.5和10 μg抗原剂量下, Typhax诱导产生的抗体水平与FDA已批准的商用伤寒疫苗Typhim Vi (25 μg) 相当^[18,85]。

6　基于非天然氨基酸的点击化学偶联技术

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合成生物学领域的非天然氨基酸插入技术, 也为多糖和载体蛋白的偶联带来了全新的可能性^[86]。该技术通过遗传密码扩展策略, 在载体蛋白的特定位点精准插入含有生物正交反应基团的非天然氨基酸, 随后利用点击化学反应实现多糖与载体蛋白的位点特异性偶联。该方法避免了传统随机连接导致的载体蛋白失活或免疫原性降低, 提高了疫苗的均一性^[87,88]。例如, Vaxcyte公司基于无细胞蛋白质合成平台开发的新型工程化载体蛋白eCRM, 通过定点引入叠氮苯丙氨酸 (pAzF) 作为点击化学偶联位点, 利用叠氮-炔烃环加成反应实现与肺炎链球菌荚膜多糖的定点偶联, 成功构建了24价肺炎链球菌结合疫苗VAX-24。目前, 该疫苗已完成Ⅱ期临床试验, 显示出与当前标准疫苗Prevnar 20 相当的安全性和耐受性^[16,89,90]。Vaxcyte公司的这一技术体系也获得FDA的突破性疗法认定, 展现了成为下一代Best-in-Class肺炎链球菌结合疫苗的潜力。可以认为, 基于非天然氨基酸的点击化学偶联技术为多糖结合疫苗的制备提供了高效、可控的新工具。

7　展望

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多糖结合疫苗被认为是目前成功的细菌性疫苗形式之一, 它在传染病防控方面发挥了巨大作用。在新兴的多糖结合疫苗的生物合成领域, 人工智能凭借其在蛋白质设计方面展现出的革命性潜力, 将为载体蛋白和OST的设计与优化提供前所未有的技术支撑^[91-95]。一方面, 人工智能在现有蛋白质改造中发挥着关键作用。对于载体蛋白而言, 人工智能可以通过优化其免疫原性、稳定性和靶向性来提高疫苗的免疫效果; 对于OSTase而言, 人工智能可以通过提升其催化效率和底物特异性来扩大其应用范围。另一方面, 人工智能驱动的从头设计, 有望设计出自然界中不存在的载体蛋白和OSTase, 为多糖结合疫苗的生产提供全新的工具, 例如, 可用来创造天然不存在的蛋白纳米颗粒作为疫苗载体^[91,93,94]。在未来, 人工智能将会重塑多糖结合疫苗的研发范式并大幅提升开发速度, 从而更好地应对新发传染病的挑战。

传统的多糖结合疫苗接种方式以肌肉注射或皮下注射为主, 尽管其免疫效果稳定且技术成熟, 但仍存在冷链依赖、接种依从性低等局限性。近年来, 新型接种方式 (如微针贴片、吸入式疫苗) 的兴起, 在无痛化、便捷性、免疫效果及成本效益等方面展现出显著优势, 为突破传统疫苗分发的瓶颈提供了新的解决方案。未来, 随着技术的进一步成熟和政策支持的加强, 新型接种方式有望显著提高全球疫苗接种覆盖率, 为传染病防控和公共卫生平等注入新动力。

此外, 多糖结合疫苗的生产技术也正在从传统的细胞生产方式向无细胞生产方式拓展。无细胞系统凭借快速、精准、低成本等优势, 为疫苗研发带来了新机遇。值得注意的是, 该技术将与PGCT、GMMA及合成糖技术等新一代技术形成互补而非替代关系, 其他新技术仍不可或缺。展望未来, 通过整合多种先进技术平台开发新型“多联多价”多糖结合疫苗, 将成为疫苗研发领域的另一个重要发展方向。

参考文献

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2025年第60卷第7期

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doi: 10.16438/j.0513-4870.2025-0353

接收时间：2025-03-29
首发时间：2026-09-20
出版时间：2025-07-12

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收稿日期：2025-03-29
修回日期：2025-05-14

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军事科学院军事医学研究院, 病原微生物生物安全全国重点实验室, 北京 100071

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王恒樑, E-mail: wanghl@bmi.ac.cn

朱力, E-mail: jewly54@bmi.ac.cn

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2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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Pathogen	Polysaccharide	Non-sugar moiety	Phase of clinical trial	Company	Technical type	Ref.
Shigella	O-polysaccharides of S. flexneri 2a	rEPA	Phase Ⅱb NCT02646371	Limmatech Biologics	PGCT	[8]
O-polysaccharide of S. dysenteriae 1	rEPA	Phase Ⅰ NCT01069471	Limmatech Biologics	PGCT	[9]
O-polysaccharide of S. sonnei	OMV	Phase Ⅱb NCT03527173	GSK	GMMA	[10]
O-polysaccharides of S. flexneri 2a, 3a, and 6, and S. sonnei	rEPA	Phase Ⅱb NCT06615375	Limmatech Biologics	PGCT	[11]
O-polysaccharides of S. flexneri 2a	TT	Phase Ⅱ NCT04602975	Institute Pasteur	Semi-synthetic glycoconjugate vaccines	[12]
O-polysaccharides of S. flexneri 1b, 2a, and 3a and S. sonnei	OMV	Phase Ⅱ NCT06663436	GSK	GMMA	[13]
Streptococcus pneumoniae	Capsular polysaccharide of serotype 12F	rEPA	Phase Ⅰ NCT03303976	Limmatech Biologics	PGCT	[14]
Capsular polysaccharides of 24 serotypes	CP1 fusion protein	Phase Ⅱ NCT03803202	Affinivax	Multiple antigen presenting system	[15]
Capsular polysaccharides of 24 serotypes	eCRM197	Phase Ⅱ NCT05297578	Vaxcyte	Click chemistry-based conjugation via non- natural amino acids	[16]
Salmonella	O-polysaccharides of S. typhimurium and Salmonella enteritidis	OMV	Phase Ⅰ/Ⅱa NCT05480800	GSK	GMMA	[17]
Vi-capsular polysaccharide of S. Typhi	CRM197	Phase Ⅰ NCT03926455	Matrivax	Protein capsular matrix vaccine	[18]
Extraintestinal pathogenic Escherichia coli	O-polysaccharides of serotypes 1A, 2, 6A, and 25B	rEPA	Phase Ⅱ NCT02546960	Janssen Research & Development, LLC	PGCT	[19]
O-polysaccharides of 9 serotypes	rEPA	Phase Ⅲ NCT04899336	Janssen Research & Development, LLC	PGCT	[20]
O-polysaccharides of 10 serotypes	rEPA	Phase Ⅰ/Ⅰia NCT03819049	Janssen Research & Development, LLC	PGCT	[21]

Pathogen

Polysaccharide

Non-sugar moiety

Phase of
clinical trial

Company

Technical type

Ref.

Shigella

O-polysaccharides of S. flexneri 2a

rEPA

Phase Ⅱb
NCT02646371

Limmatech Biologics

PGCT

[8]

O-polysaccharide of S. dysenteriae 1

rEPA

Phase Ⅰ
NCT01069471

Limmatech Biologics

PGCT

[9]

O-polysaccharide of S. sonnei

OMV

Phase Ⅱb
NCT03527173

GSK

GMMA

[10]

O-polysaccharides of S. flexneri
2a, 3a, and 6, and S. sonnei

rEPA

Phase Ⅱb
NCT06615375

Limmatech Biologics

PGCT

[11]

O-polysaccharides of S. flexneri 2a

Phase Ⅱ
NCT04602975

Institute Pasteur

Semi-synthetic
glycoconjugate
vaccines

[12]

O-polysaccharides of S. flexneri
1b, 2a, and 3a and S. sonnei

OMV

Phase Ⅱ
NCT06663436

GSK

GMMA

[13]

Streptococcus
pneumoniae

Capsular polysaccharide of
serotype 12F

rEPA

Phase Ⅰ
NCT03303976

Limmatech Biologics

PGCT

[14]

Capsular polysaccharides of 24
serotypes

CP1
fusion protein

Phase Ⅱ
NCT03803202

Affinivax

Multiple antigen
presenting system

[15]

Capsular polysaccharides of 24
serotypes

eCRM197

Phase Ⅱ
NCT05297578

Vaxcyte

Click chemistry-based
conjugation via non-
natural amino acids

[16]

Salmonella

O-polysaccharides of S. typhimurium
and Salmonella enteritidis

OMV

Phase Ⅰ/Ⅱa
NCT05480800

GSK

GMMA

[17]

Vi-capsular polysaccharide of
S. Typhi

CRM197

Phase Ⅰ
NCT03926455

Matrivax

Protein capsular
matrix vaccine

[18]

Extraintestinal
pathogenic
Escherichia coli

O-polysaccharides of serotypes
1A, 2, 6A, and 25B

rEPA

Phase Ⅱ
NCT02546960

Janssen Research &
Development, LLC

PGCT

[19]

O-polysaccharides of 9 serotypes

rEPA

Phase Ⅲ
NCT04899336

Janssen Research &
Development, LLC

PGCT

[20]

O-polysaccharides of 10 serotypes

rEPA

Phase Ⅰ/Ⅰia
NCT03819049

Janssen Research &
Development, LLC

PGCT

[21]