COVID-19 S-Trimer vaccine (Sichuan Clover Biopharmaceuticals, Inc)

点击上方图片链接可报名疫苗佐剂（Vaccine adjuvants）被定义为与疫苗抗原联合给药时能够增强疫苗免疫反应的强度、广度和持久性的各种成分，被广泛用于疫苗开发，是大部分疫苗产品不可缺少的组成部分。佐剂的范围可以涵盖从合成小分子化合物到复杂的天然提取物和纳米颗粒材料。常见的有矿物盐，微生物产物，乳液，皂苷，合成小分子激动剂，聚合物，纳米颗粒和脂质体。尽管佐剂长期以来一直广泛用于疫苗中，但它们增强免疫反应的机制尚未得到很好的表征，因此对佐剂的作用机制几乎没有系统的概括和总结。此外，现已有或正处于研发阶段的佐剂平台，业内普遍对机制、特性、免疫有效性和应用场景缺乏系统和深入的了解，难以匹配和设计适合特定疫苗的佐剂。这些原因导致目前的疫苗产品在临床实践与应用中渐显颓势，如无法提供长期保护性免疫，无法为老年、易感人群提供有效的免疫保护。2023年7月19日，来自四川大学华西医院的研究团队在Signal Transduction and Targeted Therapy上发表了题为“Vaccine adjuvants: mechanisms and platforms”的综述文章，在本文中详细解读了疫苗佐剂增强疫苗效力的作用机制，并对当前已有的佐剂平台（含在研）进行了分析汇总。疫苗佐剂的作用机制根据作用机制，佐剂通常被分为免疫刺激剂（Immunostimulants）和递送系统（Delivery Systems）两大类，但核心概念不外乎通过直接或间接帮助疫苗成分激活APC，促进抗原呈递信号（图1，①）和共刺激信号（图1，②）产生。抗原呈递信号的产生，首先由抗原被摄取和处理后呈递在APC表面的抗原肽-主要组织相容性复合体（MHC）上，并与naive T细胞表面TCR受体结合诱导T细胞的激活。共刺激信号包括表达在APC表面的共刺激分子（例如 CD40、CD80、CD86）和分泌的炎性细胞因子（例如 IL-6、IL-10、IL-12 和 TNF-α），通过与naive T细胞表面共刺激受体（CD28或细胞因子受体）结合，诱导T细胞激活，增强适应性免疫反应。图1. 佐剂的作用机制示意图免疫刺激剂免疫刺激剂通常为危险信号分子(Danger signal molecules)，以化学合成的小分子激动剂充当PAMP，DAMP，通过靶向APC上的模式识别受体(PRR)，触发先天免疫反应并诱导APC的激活和成熟。APC成熟后终止抗原吞噬活性，并增强其抗原呈递和表达高水平共刺激信号和细胞因子的能力，启动并增强适应性免疫反应。需要注意的是，不同类型的免疫刺激剂会通过不同PRR发出信号并导致不同的细胞因子分泌。目前所使用的免疫刺激剂主要按靶向受体的不同分为4类（图2）。图2. 免疫刺激剂作用机制靶向TLRs免疫刺激剂通过靶向激活TLRs（Toll 样受体），可以增强抗原呈递并上调共刺激信号和细胞因子表达（图2a）。TLR1，TLR2，TLR4，TLR5和TLR6通常在细胞表面表达，主要识别生物膜成分，包含脂质，脂蛋白和蛋白质。作用于该类TLRs的相应免疫刺激剂将促进促炎细胞因子，如IL-1β，TNF-α和IL-6的产生，并最终激活Th1或Th2型免疫应答。TLR3、TLR7、TLR8和TLR9是在细胞囊泡中表达并与核酸反应的细胞内TLRs。相应类型的免疫刺激剂激活这些细胞内TLR通常会导致I型干扰素的产生。27I型干扰素可促进Th1细胞分化并调节其功能。此外，I型干扰素通过常规树突状细胞（DC）促进抗原交叉呈递到CD8 T细胞，并直接刺激CD8 T细胞的增殖。靶向cGAS-STING通路cGAS-STING全称为环鸟苷单磷酸-腺苷单磷酸合酶-干扰素基因刺激因子，该通路在协调先天免疫和适应性免疫方面具有独特的作用，受到佐剂研发科学家们的广泛青睐（图2b）。cGAS是一种细胞质DNA受体，被双链DNA激活后将促进胞质单磷酸腺苷(Adenosine monophosphate, AMP)和鸟苷单磷酸(guanosine monophosphate, GMP)环化形成环鸟苷酸-腺苷酸(cGAMP)。随后cGAMP将与STING二聚体的V形结合域结合，引起STING的构象变化，聚集和活化，并激活NF-κB和IRF3，促进促炎细胞因子和I型干扰素表达。靶向CLRsC型凝集素受体（CLR）是一个超家族，包括Dectin-1，Dectin-2，MINCLE，DC-SIGN，CD206，CD205等，主要位于细胞膜上，作为抗原受体参与抗原捕获和抗原呈递（图2c）。具有碳水化合物结构的免疫刺激剂通常会激活CLR，随后诱导APC启动抗原的内化，加工和呈递，以增强适应性免疫应答。此外，一部分免疫刺激剂激活CLRs后可激活不同信号通路，诱导特定细胞因子的表达，控制naive T细胞的极化方向。如β-葡聚糖通过激活Dectin-1诱导Syk激酶磷酸化，进而激活NF-κB通路，促进促炎细胞因子IL12，IL-1β，IL6和IL23的表达，诱导naive T细胞分化为Th1和Th17细胞。Th1细胞诱导强烈的CTLs（细胞毒性T淋巴细胞）反应，并驱动B细胞中免疫球蛋白转化为IgG2a。Th17效应细胞的特征在于产生IL-17，IL-17F和IL-22，并通过募集中性粒细胞引发大量炎症反应。靶向其他PRRs 除上述机制外，免疫激动剂还可激活其他PRRs，主要分为核苷酸结合寡聚结构域样受体(Nucleotide-binding oligomerization domain-like receptors, NLRs) 和视黄酸诱导基因I样受体(Retinoic acid-inducible gene I-like receptors, RLRs)。核苷酸结合寡聚化结构域1(NOD1)、核苷酸结合寡聚化结构域2(NOD2)和NOD样受体热蛋白结构域相关蛋白3(NLRP3)是NLRs家族的主要成员。免疫刺激剂通过激活该类受体可诱导APC上调MHC II，增强抗原呈递。此外，该类受体的激活将导致促炎细胞因子如IL-1β和IL-18的表达，驱动naive T细胞偏向极化为Th2型细胞，产生Th2细胞免疫反应。视黄酸诱导基因I(RIG-I)和黑色素瘤分化相关基因5(MDA5)是RLRs家族的主要成员，主要识别RNA。激活RIG-I和MDA5将导致IRF3和IRF7的激活，进而诱导I型干扰素的表达。靶向这两种受体的免疫刺激剂最终偏向于导致Th1细胞和CTL的产生。递送系统递送系统通常被定义为搭载抗原以增加APCs对抗原摄取和呈递的载体材料。主要促进抗原的呈递，包括APCs对抗原的识别、摄取和内化。因此，递送系统通常可以以一种或多种方式增强抗原呈递信号（图3）。图3. 递送系统作用机制延长抗原生物利用度目前延长抗原生物利用度的策略主要有（1）允许抗原持续释放；（2）形成免疫生态位；（3）为抗原载物提供保护延长生物利用度，使APCs有足够抗原促进MHC抗原肽信号产生。抗原持续释放目前已被证实可实现抗原持续释放的递送系统，包括纳米乳液递送系统(MF59, AS03)，可注射水凝胶以及可生物降解的PLGA或聚酐聚合物制成的固体聚合物颗粒。此外，具有大孔结构的自组装支架也具有缓慢释放抗原的能力，且还可同时搭载免疫刺激剂来释放炎症信号，提高血清抗体水平和CTL水平。形成免疫生态位一些递送系统可以通过在注射部位形成抗原库/免疫生态位来实现更高效的免疫活化。除了延长抗原在免疫系统中的作用时间，还可招募更多的免疫细胞浸润注射部位，这将增加抗原的摄取并提供局部炎症线索来激活随后的适应性免疫反应。先天免疫细胞的浸润会进一步使得生态位形成递送材料比常规递送材料具有更强大的免疫激活能力，并可掺入细胞因子，增加细胞迁移。可注射聚己内酯-聚乙二醇-聚己内酯热敏水凝胶可同时包封GM-CSF和卵清蛋白，增加注射部位DCs的募集，并提高抗原摄取效率。介孔二氧化硅棒是一种具有自佐剂效应的递送材料，可以激活NLRP3先天免疫路径。当注射到小鼠体内时，它会增加先天免疫细胞对注射部位的浸润并增强免疫反应。为抗原载物提供保护递送系统可以通过保护以DNA和mRNA为主的抗原免受体内酶的分解来延长抗原的生物利用度，从而保持其生物活性。mRNA抗原容易被细胞外血清中的RNA酶降解。LNP是一种经过验证的有效mRNA递送系统，其基本功能之一是封装mRNA并保护其免受降解RNA酶的影响。增强APCs靶向性 递送系统可通过两种主要方式增强APCs靶向性。第一种方法是通过模拟病原体的大小、维度和空间结构，利用微尺度或纳米级材料来促进APC摄取抗原。与可溶性抗原相比，用脂质体纳米颗粒包封抗原可改善APC对抗原的识别和内吞作用，从而增加抗原呈递和诱导更高幅度和更高亲和力的抗体。此外，免疫系统会以高度敏感的方式识别和响应病原体固有的高度有序和重复的空间结构，自组装蛋白质纳米颗粒和其他多价颗粒递送系统通过模拟病原体的这类结构可有效增加APCs抗原摄取率。值得注意的是，即使没有补充其他免疫刺激剂，这些递送系统也能引起强烈的体液和细胞免疫反应。递送系统高度有序和重复的空间结构也有利于B细胞受体（BCR）的共聚集，导致B细胞的强激活。第二种方法是通过直接靶向APCs表面特定受体（Fc受体，CLR）增加抗原摄取。纳米颗粒递送系统的表面可以被特异性修饰以主动靶向特定的细胞群。使用CLRs配体（通常是碳水化合物，如甘露糖）来修饰递送系统将导致抗原更直接地靶向特异性DC，从而增强抗原摄取。淋巴结递送由于淋巴结是初始免疫反应的部位，聚集大量先天免疫细胞和淋巴细胞，因此成为递送系统的关键靶点。常用方法通过设计合适尺寸和表面特性（如电荷和疏水性）可以使递送载体通过被动扩散进入淋巴管和淋巴结。据报道，最佳尺寸在通常在5到100 nm之间，若过小就会进入毛细血管而太大就难以通过淋巴管内皮细胞间隙。在该种方法中，递送材料通常设计为携带负电荷，避免与淋巴管间质基质中带负电荷的糖胺聚糖相互作用降低通过效率。另一种方式被称为内源性白蛋白“搭便车”机制。内源性白蛋白是脂肪酸分子的转运蛋白，利用其在淋巴系统中的循环特性，将抗原与内源性白蛋白结合，可实现淋巴结的靶向。早前报道的一项具有亲脂性尾巴的肿瘤肽疫苗通过结合白蛋白在淋巴结中大量积累，小鼠注射后使T细胞产量增加30倍，抗肿瘤功效显著增强。抗原的交叉呈递抗原的交叉呈递是外源性抗原通过MHC I分子呈递给CD8 T细胞的过程，也是抗病毒感染疫苗和癌症疫苗设计中的重要一步。在正常情况下，外源性抗原大多仅由APC单独内化，并由MHC II分子呈递给CD4 T细胞，不会发生任何交叉呈递。研究已报道三种主要方式：质子海绵效应当具有质子胺基团的阳离子聚合物或脂质递送载体被APC内化后将缓冲内涵体/溶酶体的酸性环境，导致大量氯离子和细胞质的水流入使得内涵体/溶酶体肿胀破裂，释放抗原并促进MHC I分子抗原的交叉呈递。内涵体/溶酶体膜融合一些递送系统通过融合或结合内涵体/溶酶体膜来破坏稳定性，促进抗原释放。颗粒化乳液(PAPE)就可通过结合内涵体/溶酶体膜，破环其稳定性，使抗原释放到细胞质中并交叉呈递给CD8 T细胞。光化学内化释放技术将光敏剂掺入递送系统中和抗原一起递送，光敏剂暴露于特定光源后会被激发形成单线态氧(singlet oxygen)，导致脂质过氧化和内涵体膜破坏，使抗原释放到细胞质中，增强CD8 T细胞介导的免疫应答。经典佐剂平台铝佐剂，MF59，AS01，AS03，AS04和CpG ODN 1018是经典的人用疫苗佐剂。它们已被广泛批准用于各种疫苗，并用于增加疫苗抗体滴度和增强细胞免疫反应。铝佐剂铝佐剂是第一类获准用于人用疫苗的佐剂，上市疫苗中常用的两种铝基佐剂是氢氧化铝和磷酸铝，具有广泛认可的安全性和可靠性。铝佐剂通过促进Th1细胞反应来增强IgG2和IgE抗体的产生。然而，铝助剂的作用机制很复杂，学术界仍在争论，但已在延长抗原的生物利用度和激活先天免疫途径的PRR这两个机制上得到了充分的认识。铝佐剂可以充当与抗原紧密结合并持续释放抗原的递送系统延长抗原的生物利用度并增加抗原呈递。此外，铝佐剂也可作为免疫刺激剂诱导DAMPs的产生，从而激活先天免疫途径的PRR，从而产生IL-1β和Th2型免疫应答等细胞因子。有研究表明，铝佐剂可在注射部位诱导宿主细胞死亡并释放宿主DNA或尿酸充当内源性危险信号，作为DAMPs诱导先天免疫途径的激活。目前学术界的争论点在于铝佐剂靶向的PRR，但并不影响铝佐剂成为疫苗行业的首选佐剂。含铝佐剂广泛用于预防和治疗各种疾病，包括白喉、破伤风、脑膜炎和乙型肝炎病毒（HBV）疫苗，已获得美国食品药品监督管理局（FDA）的批准。然而，铝佐剂也有一些缺点，包括难以诱导强烈的细胞免疫反应和引起不良反应（红斑，过敏反应）的可能性。目前佐剂研究科学家致力于改进配方和开发纳米铝佐剂来克服现有的缺点。 图4. 铝佐剂激活的信号通路乳佐剂MF59由角鲨烯、吐温80和司盘85组成。MF59于1997年获准作为流感疫苗佐剂，成为第一个获准用于人用疫苗的非铝佐剂。MF59乳剂具有抗原递送和免疫刺激的双重功能。MF59可用作乳液递送系统，当与抗原共同递送时，它可以延长抗原与免疫系统的相互作用，并通过在淋巴结中缓慢释放抗原来增加抗原呈递，可激活更多的APC抗原信号呈递。此外，MF59还充当免疫刺激剂。MF59可以靶向特定的PRR，并通过诱导产生内源性危险信号激活先天免疫细胞。在肌肉中施用MF59可激活先天免疫细胞，如巨噬细胞和DC，并促进趋化因子如CC-趋化因子配体2（CCL2），CCL4，CCL5和CXC-趋化因子配体8（CXCL8）的产生。这些趋化因子又可以将更多的先天免疫细胞募集到注射部位以进一步放大免疫反应，并促进这些招募的先天免疫细胞迁移到引流淋巴结以激活B细胞和T细胞。然而，MF59的确切靶向PRR尚不清楚，一般认为MF59可能通过NLRP3非依赖性ASC激活途径和TLR非依赖性MyD88激活途径起作用。简而言之，接种MF59佐剂会导致Th2偏向的免疫反应和体内Th1反应的弱诱导。目前，MF59已广泛应用于多种人用疫苗中，并显示出良好的安全性和有效性。AS03由α-生育酚、角鲨烯和吐温80组成，和MF59佐剂作用相似。与MF59一样，AS03通过NLRP3非依赖性ASC激活途径和TLR非依赖性MyD88激活途径发挥免疫刺激作用。此外，AS03还含有α-生育酚作为额外的免疫刺激剂成分。研究表明，α-生育酚参与调节某些趋化因子和细胞因子的表达，如CCL2、CCL3、白细胞介素6和CXCL1，可增强APC的抗原摄取，增加募集到引流淋巴结的先天免疫细胞的数量。一般来说，AS03也主要诱导Th2偏向的免疫反应，对Th1反应的诱导较弱。AS03主要用于多种流感疫苗中，以增加抗体的滴度和持久性。最近，AS03在COVID-19疫苗（NCT04450004，NCT04405908）的开发中也显示出良好的临床益处。基于TLR激动剂分子的佐剂AS04是通过铝吸附TLR4激动剂分子制备的，含有铝佐剂，因此还具有免疫刺激和传递抗原的功能。但AS04的免疫刺激功能强于铝佐剂，这是因为AS04含有一种更有效的免疫刺激分子，称为单磷酰脂质A（MPLA）。MPLA是从LPS中分离出的低毒性衍生物，可以特异性激活APC上的TLR4，导致NK-κB的激活和促炎细胞因子的表达，从而产生更强的Th1细胞反应。此外，当将补充AS04的疫苗与仅补充铝佐剂的疫苗进行比较时，发现补充AS04的疫苗诱导了更高水平的抗体。这证明了TLR4激动剂MPLA在AS04中起到重要作用。总体而言，AS04可以诱导比铝佐剂更强的抗体反应和Th1细胞反应，并诱导更平衡的Th1 / Th2免疫反应。目前被批准主要用于人瘤病毒（HPV）疫苗（Cervarix）和HBV疫苗（Fendrix），增加抗体产生和抗原特异性T细胞的活化。CpG ODN 1018是一种人工合成的单链DNA分子，作为TLR激动剂已被广泛研究。CpG ODN 1018特异性激活TLR9，并导致TRF7的激活，进而导致促炎细胞因子和I型干扰素的表达，最终引起强烈的Th1型细胞反应和细胞毒性T细胞产生。CpG ODN 1018最初被批准用于HBV疫苗，目前正在临床试验中作为COVID-19疫苗（NCT04450004，NCT04405908）的潜在疫苗佐剂进行评估。三叶草生物SCB-2019的CpG ODN 1018佐剂COVID-19疫苗已经在国内获紧急使用授权。微粒佐剂系统AS01是一种经典的脂质体微粒佐剂系统，含有免疫刺激剂MPLA和从Quillaja Saponaria(QS)树皮中提取的称为QS-21的活性成分，具有抗原呈递和免疫刺激的双重功能。该脂质体是一种中空磷脂双层人造膜，可用于抗原的包封和递送，保护抗原不被降解并延长其生物利用度，从而使APC能够捕获更多的抗原信号。AS01的MPLA通过TLR4激活先天免疫系统，促进Th1型反应增加。QS-21激活APC中的NLRP3，随后激活半胱天冬酶1以促进活性细胞因子IL1β，IL18和IL33的表达。此外，还发现QS-21可促进内涵体逃逸并促进交叉呈递。总体而言，这两种免疫刺激剂成分协同作用，使AS01诱导以Th1为主的免疫反应并促进CTL的产生。AS01是已获得许可的疟疾和带状疱疹疫苗的一部分。最近，AS01还被应用于开发针对结核病的新型肽疫苗（M72 / AS01E），并显示出在健康成人中预防结核病的安全性和有效性前景（NCT01755598，NCT01755598）。研究中的佐剂平台目前被广泛使用的佐剂在增强疫苗效力的同时也暴露了一些问题。例如，它们增强疫苗免疫力的能力较弱，不提供长期保护性免疫力，对老年人群效果较差。此外，这些批准的佐剂大多只能诱导抗体反应，诱导CD8 T细胞介导的细胞免疫能力较弱，这对于针对病毒感染性疾病和癌症疫苗至关重要。大多数许可佐剂的作用机制在很大程度上仍不清楚，这可能导致这些佐剂的不当使用和无法控制的副作用。因此，需要新型佐剂来解决这些问题。双链核糖核酸 （dsRNA）人工体外合成的双链RNA（dsRNA）可以靶向并激活APC上的TLR3和MDA5，导致促炎细胞因子如IL-12和I型干扰素的产生，并促进强Th1极化免疫反应和CTL的产生。Poly-I：C及其修饰的变体Poly-ICLC是研究最多的合成dsRNA免疫刺激剂。在体外实验中，poly-I：C和poly-ICLC诱导人外周血单核细胞来源DC的成熟，促进IFN-β和促炎细胞因子IL-6和IL-12的分泌，使外源性抗原与CD8 T细胞发生交叉呈递并触发Th1极化免疫反应。poly-I：C和poly-ICLC在临床上多用于肽疫苗，DC疫苗和全细胞疫苗，主要用于多种癌症疫苗的开发。在癌症疫苗中添加poly-I：C或poly-ICLC可增强疫苗诱导的抗肿瘤T细胞和NK细胞反应，从而有助于肿瘤消退或根除。然而，值得注意的是，poly-I：C/poly-ICLC 除了具有触发强大免疫反应的功能外，还可能具有剂量依赖性全身发热和凝血异常等副作用。因此，应考虑合理的给药系统以保护其免受降解，在剂量上需要确保激活APCs并减少副作用。图5. 目前用于癌症疫苗开发的poly-I：C/poly-ICLCGLA及其衍生物吡喃葡萄糖脂质A（GLA）是一种人工合成的LPS模拟物，是MPLA的替代品，通过激活APC上的TLR4在体内诱导Th1型免疫反应。GLA通常制备为吡喃葡萄糖基脂质A-水性纳米悬浮液（GLA-AF），吡喃葡萄糖基脂质A-稳定乳液（GLA-SE），吡喃葡萄糖脂质A-脂质体（GLA-LS）和吡喃葡萄糖脂质A-氢氧化铝（GLA-明矾），以GLA-SE在临床前和临床研究中研究最多。在几项临床试验中，当GLA-SE被用作结核病疫苗的佐剂并针对ID93重组蛋白进行测试时，发现该疫苗可诱导高滴度抗原特异性抗体产生和CD4 T反应，在接种疫苗的人群中显示出良好的安全性和免疫原性（NCT01599897，NCT02465216，NCT02508376，NCT01927159）。咪唑喹啉咪唑喹啉能够激活TLR 7/8通路，激活NF-κB和IRF7，促进促炎细胞因子和I型干扰素的表达，引起强大的Th1细胞免疫反应和CTLs产生。咪喹莫特（R837）、瑞西莫特（R848）和3M-052（也称为替拉莫德）是临床前和临床环境中研究最多的咪唑喹啉佐剂。大量证据表明，R837在黑色素瘤、HPV、乳腺癌和T细胞淋巴瘤疫苗中表现出很强的佐剂作用。病毒疫苗和肿瘤疫苗与R848相结合已被证明可诱导更强的体液反应和CD8 T细胞免疫反应。然而，咪唑喹啉作为疫苗佐剂仍存在一些缺点。具体而言，它们经常从注射部位扩散至全身，从而远离抗原，降低疗效并诱发全身副作用。因此，这些咪唑喹啉佐剂需要与合成聚合物支架、纳米凝胶、脂质基纳米颗粒或其他递送材料偶联，以增强其免疫作用并减少其副作用。环状二核苷酸 （CDN）CDN可以靶向并激活cGAS-STING途径，导致IRF3和NF-κB的激活，诱导I型干扰素和促炎细胞因子的产生，刺激抗原交叉呈递并产生强烈的Th1型细胞响应和CTL响应。目前还未有使用这些合成CDN作为疫苗佐剂的具体例子。由于这些合成CDN在临床前和临床试验中已显示出显着增强人体免疫反应的能力，我们相信使用这些合成CDN作为疫苗佐剂的愿望将很快实现（NCT03172936，NCT03010176，NCT03010176，NCT04096638，NCT04020185），利用纳米颗粒包封CDNs被视为提高CDNs体内利用效率的策略。代谢佐剂 一些靶向代谢途径的小分子调节剂被发现可用作新型疫苗佐剂。靶向甲羟戊酸途径的小分子抑制剂在增加小鼠和食蟹猴的抗体滴度和增强细胞免疫反应方面显示出佐剂作用，延长抗原保留从而增强了抗原呈递。许多研究结果表明，细胞代谢途径的酶或调节因子可能是设计和开发新型佐剂的潜在靶标。锰佐剂及其衍生物锰（Mn）及其衍生物被发现具有潜在的佐剂活性，可充当cGAS的激活剂，诱导2'3'-cGAMP的非规范催化合成，并通过这种方式激活cGAS-STING的先天免疫途径，诱导I型干扰素的产生。研究人员开发了一系列基于Mn纳米助剂，例如纳米级锰凝胶（MnJ）。它不仅具有激活cGAS-STING通路作为免疫刺激剂诱导I型干扰素产生的能力，而且还具有携带抗原的递送系统作用。研究中的递送系统平台 脂质纳米颗粒LNP 是非病毒纳米脂质囊泡递送系统，由可电离脂质、磷脂、胆固醇和聚乙二醇修饰脂质组成。可电离脂质是LNPs的主要成分，磷脂和胆固醇有助于LNPs的结构完整性，聚乙二醇改性有助于维持LNPs的稳定性。LNP的作用机制主要包括（1）为抗原提供有效的货物保护，延长其生物利用度;（2）增加抗原的粒径以靶向APCs并促进APCs吸收;（3）通过膜融合促进抗原从内体逃逸，引起CD8 T细胞免疫应答。LNP已被用于各种疫苗的递送，也被认为是目前最适合mRNA疫苗的递送系统聚合物颗粒聚合物颗粒一般分为天然聚合物颗粒和合成聚合物颗粒，主要实现抗原的缓慢释放。疫苗递送中最常用的天然聚合物是壳聚糖。壳聚糖具有高阳离子电荷和生物粘附特性。因此，壳聚糖可以通过静电相互作用与阴离子核酸形成紧密的络合物。此外，壳聚糖的生物粘附特性使其能够长时间与粘膜表面保持接触，促使抗原持续刺激免疫细胞，表明壳聚糖可能是核酸疫苗和粘膜疫苗的有前途的递送系统。此外，壳聚糖最近还被发现通过激活cGAS-STING通路诱导CD8 T细胞免疫的I型干扰素产生。与天然聚合物相比，合成聚合物颗粒通常具有更高的重现性和更可控的缓释速率。PLGA是一种合成聚合物颗粒材料，现在被广泛用作疫苗递送的载体。目前，PLGA颗粒作为癌症疫苗的输送系统已经经过临床测试（NCT01753089，NCT04751786）。此外，PLGA已被FDA批准用于兽用药物递送。因此，相信基于PLGA的疫苗递送系统将在不久的将来被批准用于人类疫苗。病毒样颗粒 （VLP）VLP来源于病毒衣壳的外壳蛋白，由蛋白质单体的自组装形成，其出色的粒径和几何形状使其成为递送疫苗抗原的有效平台，使疫苗很容易进入淋巴管，靶向淋巴结。由于VLPs的高度有序和重复的空间结构，非常有利于交联BCR，即使在没有T辅助（Th）细胞的情况下也能强烈激活B细胞。目前，基于VLP的疫苗已经取得成功，并且已经在市场上广泛使用，例如用于HPV的Cervarix®和Gardasil®，用于肝炎病毒的Sci-B-Vac™以及RTS,S VLP疫苗。蛋白质纳米颗粒笼（Caged protein nanoparticles）笼状蛋白质纳米颗粒通常由一系列重复的基序组成，这些基序允许抗原表位或抗原掺入其亚基结构中，从而使这些掺入的抗原表位或抗原显示在组装颗粒的表面上。与VLP类似，笼养蛋白质纳米颗粒具有高度有序和重复的空间结构以及淋巴结运输的最佳大小等特性。然而与VLP不同，笼状蛋白质纳米颗粒是非病毒来源的。蛋白质纳米颗粒中，铁蛋白应用最频繁。铁蛋白是一种具有八面体对称结构的蛋白质，由24个亚基自组装形成，每个亚基由四个α-螺旋束组成。无机纳米材料金纳米颗粒是常用的无机纳米材料输送系统之一。由于金纳米颗粒的大小、形态、疏水性、表面电荷、均匀性和分布等理化性质可以通过材料合成和表面化学修饰来精确调节，以形成特定的免疫类型，因此近年来金纳米粒子作为一种有前途的抗原递送系统获得了广泛关注。金纳米颗粒通过（1）保护抗原免受降解和（2）形成合理粒径的纳米疫苗以促进抗原直接转运到淋巴结来促进抗原呈递并增强适应性免疫反应。有研究报道，金纳米颗粒可以通过激活NLRP3炎症小体和促进Th2型细胞因子的产生来增强抗原特异性抗体的产生。与金纳米颗粒类似，介孔二氧化硅纳米颗粒（MSN）也具有负载能力强和易于表面改性的特点。改良的MSN已被研究为有效的疫苗递送系统，当用作癌症疫苗递送系统时，它增加了抗原特异性CTL的产生，抑制了小鼠的肿瘤生长，提高了荷瘤小鼠的存活率。总结选择合适的佐剂来辅助抗原呈递和改善免疫反应是开发新疫苗的重要问题。在佐剂的选择上，除了需要考虑给药途径，病原体类型，抗原类型和疾病阶段外，还需要重视接种者的生物学特征，如种族、年龄、病史和遗传因素。此外，在作为疫苗产品研究时，还需要考虑佐剂的安全性和经济性。最终这些因素都将或多或少影响疫苗佐剂的有效性。撰文：三姐夫 来源：RNAScript识别微信二维码，添加小编，符合条件者即可申请加入微信群！请注明：姓名+研究方向！声明：本文旨在传递更多信息，版权归原作者所有。原创文章转载均需经过授权并注明来源，如涉及内容、版权或其他问题请时联系小编删除！文章仅代表作者个人观点，并不代表公众号立场。本公众号拥有对此声明的最终解释权！投稿邮箱：yck19876@163.com

-- Clover’s two premium respiratory vaccines approved in China (COVID-19 and seasonal influenza) are expected to drive meaningful and diversified revenues in 2023 with continued growth thereafter -- -- Clover’s COVID-19 vaccine now listed in 24 provinces and municipalities in China (representing >80% population coverage) and well-positioned to be a major player in upcoming vaccination campaigns; anticipated to receive emergency use authorization (EUA) and sign a bilateral supply agreement in at least one additional country in H1 2023 -- -- AdimFlu-S (QIS) commercial production ongoing and on-track for H2 2023 launch as the only imported quadrivalent seasonal influenza vaccine approved in mainland China in a stable and growing market -- -- On track to build a leading respiratory vaccine franchise; currently the only company in China with commercial-stage quadrivalent seasonal influenza and recommended COVID-19 vaccines, with additional catalysts anticipated in 2023 as Clover continues to expand its pipeline and commercial footprint -- SHANGHAI, China, March 29, 2023 (GLOBE NEWSWIRE) -- Clover Biopharmaceuticals, Ltd. (Clover; HKEX: 02197), a global commercial-stage biotechnology company committed to unleashing the power of innovative vaccines to save lives and improve health around the world, today announced corporate updates and financial results for the year ended December 31, 2022. “2022 was a pivotal year for Clover as we successfully transformed into a commercial-stage, vaccines-focused biotech following regulatory clearance for our COVID-19 vaccine in China,” said Joshua Liang, Chief Executive Officer and Executive Director of Clover. “In 2023, we have expanded our commercial-stage portfolio and now have two premium respiratory vaccines (COVID-19 and seasonal influenza) which are expected to drive meaningful and diversified revenues this year with continued growth thereafter. We plan to leverage our validated R&D capabilities and continue expanding our pipeline, with a focus on building a leading respiratory vaccine franchise and establishing a presence in the pediatric vaccines market, for long-term value creation.” Key Commercial Highlights & Plans COVID-19 Vaccine Commercialization: Since receiving emergency use authorization (EUA) for its adjuvanted protein-based COVID-19 vaccine, SCB-2019 (CpG 1018/Alum), in China, Clover has made significant progress in expanding market access, with SCB-2019 now commercially launched in multiple provinces and successfully listed in 24 provinces and municipalities (representing >80% population coverage). Comprehensive Global Vaccine Development: Clinical trial results released and published in 2022 consistently demonstrated that SCB-2019 elicited broad cross-neutralization across variants. Notably, in January 2022, Clover published the final efficacy results from its global Phase 2/3 study of SCB-2019 in The Lancet. Clover also announced positive data from its Phase 3 study evaluating SCB-2019 as a heterologous COVID-19 booster vaccine. Results demonstrated broad and superior cross-neutralization of all Omicron subvariants tested, including recent major subvariants such as BQ.1.1 and XBB.1.5, in participants who received SCB-2019 as a heterologous fourth dose after three doses of inactivated vaccine compared to a fourth dose of inactivated vaccine. EUA and Expanding Market Access in China: In December 2022, SCB-2019 was included for EUA in China and subsequently recommended as a prioritized vaccine in China’s national immunization plan for a second booster dose (fourth vaccination dose) campaign targeting older adults, immunocompromised individuals and individuals with comorbidities. Since the initial launch in February 2023, Clover has commercialized its vaccine in multiple provinces and has successfully listed in 24 total provinces and municipalities (representing >80% population coverage). This broad nationwide market access positions Clover to be a major player in upcoming vaccination campaigns in 2023. With the Chinese population’s recent increasing awareness of the potential disease severity and impact of COVID-19, Clover believes that a robust annual booster market for COVID-19 vaccines in a private market setting could emerge—similar to the growing seasonal influenza vaccination market—favoring premium products such as Clover’s adjuvanted protein-based COVID-19 vaccines and introducing attractive product pricing dynamics. Ex-China Opportunities: Clover is prioritizing global (ex-China) regulatory submissions directly in select countries, primarily in Asia Pacific and Latin America, based on the potential to generate significant near-term revenue and impact via bilateral supply agreements. Clover expects SCB-2019 to receive an EUA in at least one additional country and to complete multiple EUA submissions during H1 2023, potentially driving revenue via bilateral agreements starting in 2023. In addition, Clover expects to establish at least one bilateral supply agreement in H1 2023, which could begin to drive commercial value in 2023. Manufacturing Milestones: In September 2022, Clover’s contract development manufacturing organization (CDMO) facility received a European Union Good Manufacturing Practice (EU GMP) certificate for the production of SCB-2019. Subsequently, Clover’s in-house manufacturing facility in Changxing, Zhejiang province was inspected and achieved commercial GMP status in China. With two commercial-ready, GMP-certified facilities, Clover can flexibly meet demand as it continues rolling out the vaccine. Stockpiled inventory of key raw materials to date enables Clover to potentially produce and release over 100 million doses of SCB-2019 in 2023. Quadrivalent Seasonal Influenza Vaccine Upcoming Commercialization: In February 2023, Clover announced that it entered into an exclusive agreement with Adimmune Corporation (Adimmune) to distribute AdimFlu-S (QIS) in mainland China, where it is the only imported seasonal quadrivalent influenza vaccine approved for use in individuals aged three years and older. With this deal, Clover became the only Chinese company with commercial-stage quadrivalent seasonal influenza and recommended COVID-19 vaccines and established a presence in a rapidly growing market. Market Opportunity: The market for influenza vaccine in China grew by about 35% annually1 before the pandemic and is expected to continue growing in the post-pandemic era with increasing vaccine awareness and favorable government policies. Moreover, demand in China continues to shift from trivalent to seasonal quadrivalent influenza vaccine options, which accounted for a majority of doses (70%) in 2022.2 Commercialization Plans: Adimmune has already started production of AdimFlu-S (QIS) and is on track to support a commercial launch in mainland China in H2 2023. Sales are expected to be accretive to Clover’s earnings starting in 2023 and contribute meaningful growth in 2024 and beyond. Further, the deal enables Clover to leverage its existing and growing commercial presence in China to commercialize both COVID-19 and influenza vaccines. Additional Opportunities: The agreement with Adimmune also grants Clover rights to commercialize AdimFlu-S (QIS) in Bangladesh, Brazil and the Philippines, contingent upon regulatory approvals, and to potentially collaborate with Adimmune on the development of additional vaccine candidates including next-generation influenza vaccines. Key Research and Development (R&D) Highlights & Plans Focused on Building a Leading Respiratory Vaccine Franchise: Clover is focused on building a leading respiratory vaccine franchise to address unmet needs in preventing serious respiratory infectious diseases and to capture related significant cross-promotion, co-administration, and long-term lifecycle management opportunities. In pursuing these varied value-creation opportunities, Clover is leveraging its synergistic capabilities in R&D, manufacturing, and commercialization. Prioritized areas include respiratory syncytial virus (RSV), pneumococcus and next-generation seasonal influenza. Additionally, Clover plans to establish a presence in the pediatric vaccine market, with enterovirus A71 (EV71) and DTaP (diphtheria, tetanus, and pertussis) being two prioritized areas of interest for Clover. Mid- to Late-Stage Pipeline Expansion: In addition to the Adimmune quadrivalent seasonal influenza deal, Clover further anticipates at least one additional in-licensing deal in 2023 to expand its mid- to late-stage pipeline (Phase 2, Phase 3, Commercial). Multivalent SARS-CoV-2 Vaccine Candidate: Clover is conducting research to develop a multivalent SARS-CoV-2 vaccine leveraging the Trimer-Tag technology platform and designed to be broadly protective against currently circulating and potential future strains of the virus. Clinical development is planned to begin in 2023, with Phase 3 immunological bridging to SCB-2019 planned to support potential regulatory approvals. Trimer-Tagged Pre-Clinical Assets: Clover continues to leverage the validated Trimer-Tag platform technology to develop innovative protein-based vaccines with ongoing pre-clinical vaccine R&D activities. Corporate & Financial Updates Cash Position: Cash and cash equivalents were RMB1,856.5 million as of December 31, 2022, compared to RMB2,835.3 million as of December 31, 2021, primarily attributable to preparations for the commercialization of SCB-2019 including a strategic procurement and stockpiling campaign for key raw material inventory and continued investment in R&D activities. Clover’s cash position is expected to support and position the company for success beyond 2023. Operating Expenses: R&D expenses were RMB1,465.3 million for 2022, compared to RMB1,826.3 million for 2021. This decrease was primarily attributable to the decrease in clinical trial expenses for late-stage clinical development of SCB-2019. The decrease was partially offset by the increase in raw materials and consumables usage and CDMO service fees mainly related to technology transfer and process validation which have been substantially completed in 2022. Administrative expenses were RMB410.2 million for 2022, compared to RMB345.7 million for 2021. This increase was primarily attributable to the annualization of personnel costs in 2022 for new hires, most of whom onboarded in mid-2021. It was offset by the decrease in third-party recruitment agency costs. Clover expects both R&D and G&A expenses in 2023 to decrease significantly compared to 2022, as the late-stage clinical development for SCB-2019 (including multiple global Phase 2/3 clinical trials) has been substantially completed, and the company continues to streamline corporate operations. Annual Loss Estimates: International Financial Reporting Standards (IFRS) net loss was RMB2,451.9 million for 2022, compared to RMB6,016.3 million for 2021. The decrease was primarily attributable to the non-cash, one-time change of RMB3,807.6 million in the fair value of convertible redeemable preferred shares as required under the IFRS for 2021. Non-IFRS adjusted loss was RMB2,356.9 million for 2022, compared to RMB2,083.5 million for 2021. Adjusted loss for the year represents the loss for the year excluding the effect brought by share-based payment expenses and certain non-cash items and non-recurring events, namely the fair value changes of convertible redeemable preferred shares. Placement: In December 2022, Clover announced that it completed the placement of a total of 128,000,000 new shares, receiving net proceeds of HK$500.5 million after deducting related fees and expenses. Clover has been using the net proceeds from the placement to expand commercialization capabilities and production capacity and extend working capital needs to strengthen Clover’s financial position and ensure Clover has adequate resources to support commercialization and sustained growth. About Clover Biopharmaceuticals Clover Biopharmaceuticals is a global commercial-stage biotechnology company committed to unleashing the power of innovative vaccines to save lives and improve health around the world. With integrated research and development, manufacturing and commercial capabilities as well as strong partnerships with organizations globally, Clover has a diverse pipeline of candidates that have the potential to meaningfully reduce the burden of vaccine-preventable diseases—and to make more diseases preventable. For more information, please visit Clover's website: and follow the company on LinkedIn and Twitter. Clover Forward-looking Statements This press release contains certain forward-looking statements and information relating to us and our subsidiaries that are based on the beliefs of our management as well as assumptions made by and information currently available to our management. When used, the words "aim," "anticipate," "believe," "could," "estimate," "expect," "going forward," "intend," "may," "might," "ought to," "plan," "potential," "predict," "project," "seek," "should," "will," "would" and the negative of these words and other similar expressions, as they relate to us or our management, are intended to identify forward-looking statements. Forward-looking statements are based on our current expectations and assumptions regarding our business, the economy and other future conditions. We give no assurance that these expectations and assumptions will prove to have been correct. Because forward-looking statements relate to the future, they are participant to inherent uncertainties, risks and changes in circumstances that are difficult to predict. Our results may differ materially from those contemplated by the forward-looking statements. They are neither statements of historical fact nor guarantees or assurances of future performance. We caution you therefore against placing undue reliance on any of these forward-looking statements. Any forward-looking statement made by us in this document speaks only as of the date on which it is made. Factors or events that could cause our actual results to differ may emerge from time to time, and it is not possible for us to predict all of them. Participant to the requirements of applicable laws, rules and regulations, we undertake no obligation to update any forward-looking statement, whether as a result of new information, future events or otherwise. All forward-looking statements contained in this document are qualified by reference to this cautionary statement. Clover Biopharmaceuticals: Albert Liao Executive Director, Corporate Communications media@cloverbiopharma.com Elaine Qiu Director, Investor Relations investors@cloverbiopharma.com 1 Compound annual growth rate (CGAR) based on influenza vaccine doses released in mainland China 2016-2017 and 2020-2021 with data from the China Centers for Disease Control (CDC) and National Institutes for Food and Drug Control. 2 Estimate based on batch release data from the China National Institutes for Food and Drug Control.