【进展中的科学】mRNA疫苗——免疫学的新纪元（第二部分）

要点

• 最近改进的mRNA疫苗，有助于增加蛋白质翻译、调节先天性和适应性免疫原性、改善递送。

• 在流感病毒、寨卡病毒、狂犬病病毒等动物模型中，mRNA疫苗已引发对传染病靶标的有效免疫。尤其近年来，人们使用了脂质包封或裸的序列优化mRNA。

• 多种方法mRNA癌症疫苗用于癌症临床试验，包括树突状细胞（DC）疫苗和各种类型的直接注入mRNA。结果显示，一些病例中出现抗原特异性T细胞反应，在某些情况下能延长患者的无病生存期。

• 治疗中需要考虑的因素和挑战包括：扩大良好生产规范（GMP） 生产、制定法规、进一步记录安全性和提高疗效。

  
  

• 未来重要的研究方向：比较和揭示各种mRNA疫苗激活的免疫路径，以此改进当前方法，并针对其他疾病靶点启动新的临床试验。

  
  

摘要

信使核糖核酸（mRNA）疫苗效能高、开发快，有潜力实现低制造成本、给药安全，因此有望替代传统疫苗。然而直到最近，由于mRNA不稳定、体内递送（in vivo delivery）低效，它的应用还很有限。这些问题，很大程度被最近的技术进步所解决，针对传染病和几种癌症的多种mRNA疫苗，在动物模型和人体实验中显现的结果令人鼓舞。本综述详细介绍mRNA疫苗以及将它推向普及治疗的未来方向和挑战。

正文提纲

一、mRNA疫苗药理学基础

二、mRNA疫苗技术的最新进展

1.mRNA翻译与稳定性的优化

2.免疫原性的调节

3.mRNA疫苗传递的进展

三、mRNA抗传染病疫苗

1.自扩增mRNA疫苗

2.树突状细胞mRNA疫苗

3.直接注入非复制mRNA疫苗

mRNA抗传染病疫苗

开发针对传染性病原体的预防性或治疗性疫苗，是遏制和预防流行病的最有效手段。然而，针对引起慢性或反复感染的病毒，传统的疫苗方法在大都未能成功生产出有效疫苗。这些具有挑战性的病毒，比如有HIV-1，单纯疱疹病毒和呼吸道合胞病毒（RSV）。此外，商业疫苗开发和批准的缓慢步伐，不足以应对急性病毒性疾病的迅速出现，2014年到2016年埃博拉和寨卡病毒暴发就说明了这一点。因此，开发更有效和多功能的疫苗至关重要。

mRNA疫苗的临床前研究带来希望，它满足理想临床疫苗多方面要求：它们在动物中显示出良好的安全性，具有多功能性和快速设计用于新出现的传染病，并且适合可扩展的良好生产规范（GMP）生产（已经由几家公司进行）。与蛋白质免疫不同，几种形式的mRNA疫苗诱导强烈的CD8+T细胞反应，这可能是由于MHC I类分子上内源性产生的抗原的有效呈递，以及有效的CD4 + T细胞反应[56,87,88]。此外，有别于DNA免疫，mRNA疫苗仅通过一次或两次低剂量免疫就显示出在动物中产生有效的中和抗体反应的能力[20,22,85]。因此，mRNA疫苗在动物模型中引发了针对多种传染因子的保护性免疫[19,20,22,56,89,90]，因此产生了极大的乐观情绪。然而，最近发表的两项用于传染病的mRNA疫苗临床试验的结果有些平庸（modest），这让人们对临床前成功转化为临床的预期更加谨慎[22,91]（下文将进一步讨论）。

用于对抗传染性病原体RNA疫苗主要有两种：自扩增或复制RNA疫苗和非复制mRNA疫苗。非复制性mRNA疫苗可以通过其递送方法进一步区分：离体加载DC、直接体内注入到各种解剖部位。如下所述，最近在这些领域发表的临床前研究数量迅速增加，其中一些已进入人体临床试验（表2）。

1.自扩增mRNA疫苗

目前使用的自我扩增mRNA（SAM）疫苗大多数基于甲病毒基因组[92]。它编码RNA复制机制的基因保持完整，但编码结构蛋白的基因被目的抗原取代。全长RNA长~9 kb，很容易通过IVT从DNA模板产生。抗原编码RNA在细胞内复制，因此可以从极小剂量的疫苗中产生大量抗原。一项早期研究报告称，在小鼠中，用编码RSV融合（F）、流感病毒血凝素（HA）或病病毒前膜和包膜（prM-E）蛋白的10μg裸SAM疫苗进行免疫，可产生抗体反应以及免受致命的病毒攻击的部分保护[93]。RNA络合剂的开发，让SAM疫苗的功效有了显著提高。仅100ng编码RSV F的RNA复制子疫苗与LNP络合，在小鼠中就能产生有效的T和B细胞免疫应答，并且在棉鼠鼻内攻击系统中引发针对RSV感染的保护性免疫应答[19]。在LNP或水包油阳离子纳米乳中编码流感病毒抗原的SAM疫苗，在雪貂中诱导了有效的免疫反应，并在小鼠中提供了对同源和异源病毒攻击的保护[94,95,96]。进一步的研究表明，该疫苗平台对多种病毒具有免疫原性，包括小鼠人巨细胞病毒（CMV）、丙型肝炎病毒和狂犬病病毒、兔子HIV-1以及恒河猴HIV-1和人CMV[50,87,97]。编码流感抗原的Replicon RNA与含壳聚糖的LNP或聚乙烯亚胺（PEI）复合，在皮下递送后引起小鼠的T细胞和B细胞免疫反应[98,99]。Chahal及其同事开发了一种递送平台，该平台由化学修饰的可电离树枝状聚合物络合到LNPs[89]。使用该平台，他们证明，肌内递送编码流感病毒、埃博拉病毒或刚地弓形虫抗原的RNA复制子可以保护小鼠免受致命感染[89]。同一小组最近证明，用编码寨卡病毒prM-E的RNA复制子以相同的方式配制的疫苗接种在小鼠中引起抗原特异性抗体和CD8+ T细胞反应[88]。最近的另一项研究报告了SAM疫苗对细菌病原体（即链球菌（A组和B组）属）的免疫原性和中等保护功效，进一步证明了该平台的多功能性[100]。

SAM疫苗的优点之一是它们产生自己的佐剂，这些佐剂的形式包括dsRNA结构、复制中间体和其他可能有助于其高效力的基序。然而，这些PAMP的内在性质，可能使得SAM疫苗的炎症特征或反应原性难以调节。此外，SAM疫苗的插入片段比无复制子基因的mRNA受到更多大小的限制，并且复制蛋白的免疫原性理论上可能限制重复使用。

2.树突状细胞mRNA疫苗

如上所述，离体树突状细胞（DC）负载来产生细胞介导的癌症免疫，这种方法广受追捧。使用这种方法开发的传染病疫苗，主要限于HIV-1治疗性疫苗：接受高效抗逆转录病毒治疗的HIV-1感染者，用编码各种HIV-1抗原的mRNA电穿的自体DC（autologous DCs）进行治疗，然后评估细胞免疫反应[101-106]。该干预被证明是安全的，并引发了抗原特异性CD4+和CD8+ T细胞反应，但没有观察到临床益处。另一项针对人类的研究，评估了健康人类志愿者和同种异体干细胞接受者的CMV pp65 mRNA负载DC疫苗接种，并报告了CMV特异性细胞免疫反应的诱导或增加（induction or expansion）[107]。

3.直接注入非复制mRNA疫苗

直接注入非复制的mRNA疫苗，是一种有吸引力的疫苗形式，特别当资源有限，因为管理起来简单而经济。尽管一项早期报告表明，用编码流感病毒核蛋白的脂质体复合物mRNA免疫在小鼠中引起CTL反应[108]，但仅在几年前才首次证明了mRNA疫苗对传染性病原体的保护性免疫反应[18]。这项开创性的工作表明，皮内施用编码各种流感病毒抗原的未复合物mRNA与鱼精蛋白复合RNA佐剂在多种动物模型中具有免疫原性，并保护小鼠免受致命的病毒攻击。

使用编码狂犬病病毒糖蛋白的基于鱼精蛋白的RNActive平台进行免疫，也诱导了对小鼠致命脑内病毒挑战的保护性免疫和猪的有效中和抗体反应[56]。在最近发表的一项开创性工作中，Alberer及其同事在101名健康志愿者中评估了这种疫苗的安全性和免疫原性[91]。受试者通过针头注射器或无针装置接受 三次80-640μg的mRNA疫苗（皮内或肌肉注射）。接种疫苗七天后，几乎所有参与者都报告了轻度至中度注射部位反应，78%的人经历了全身反应（例如发烧、头痛和发冷）。有一个严重的不良事件可能与疫苗有关：一过性和中度的贝尔麻痹病例（Bell palsy）。令人惊讶的是，针头注射器注射在98%的接受者中没有产生可检测到的中和抗体。相比之下，无针递送的诱导了不同程度的中和抗体，其中大多数所达到的峰值高于预期的保护阈值，但在长期随访的受试者中，1年后基本减弱。接受这种疫苗后动物和人类之间、两种递送途径之间的免疫原型都不同。对这两点不同的理解，将为未来使用该平台的疫苗设计提供信息。

其他传染病疫苗已经成功地利用了基于脂质或聚合物的递送系统。阳离子 1，2-二油酰氧基-3-三甲基丙烷铵（DOTAP）和二油酰磷脂酰乙醇胺 （DOPE） 脂质复合 mRNA 编码 HIV-1 gag 在小鼠皮下递送后产生抗原特异性 CD4+ 和 CD8+ T 细胞反应[109]。另外两项研究表明，PEI复合的mRNA可以有效地递送给小鼠以诱导HIV-1特异性免疫反应：皮下递送编码HIV-1 gag的mRNA引发CD4+和CD8 + T细胞反应，鼻内给药编码HIV-1包膜gp120亚基的mRNA穿过鼻上皮并在鼻腔中产生抗原特异性免疫反应[110,111]。Kranz及其同事还使用编码流感病毒HA的脂质复合mRNA，对小鼠进行了静脉内免疫，并在单次剂量后显示出T细胞活化的证据[59]。

核苷修饰的mRNA疫苗代表了一种新的、高效的mRNA疫苗类别。由于这种免疫平台的新颖性，我们对疗效的了解仅限于最近四篇发表结果，这些发表证明了，这种疫苗在小型和大型动物中的效力。第一份发表的报告表明，单次皮内注射编码寨卡病毒prM-E的LNP配方mRNA，用1-甲基假尿苷和FPLC纯化修饰，在猕猴中使用低至50μg（0.02mg kg-1）疫苗即可引起小鼠和恒河猴的保护性免疫反应[20]。另一个小组随后进行的一项研究,在小鼠中测试了一种类似设计的寨卡病毒疫苗，发现单次肌内免疫会引起中度免疫反应，而加强疫苗接种会产生有效和保护性的免疫反应[85]。该疫苗还掺入了修饰的核苷1-甲基假尿苷，但没有报告FPLC纯化或其他去除dsRNA污染物的方法。值得注意的是，该报告显示，通过去除E蛋白中的交叉反应表位，可以减少异源黄病毒继发感染的抗体依赖性增强，这是登革热和寨卡病毒疫苗的主要关注点。最近的一项后续研究在母亲疫苗接种和胎儿感染模型中评估了相同的疫苗[112]。两次免疫接种将胎儿小鼠的寨卡病毒感染率降低了几个数量级，并完全挽救了胎儿活力的缺陷。

最近的另一份报告评估了LNP复合、核苷修饰、非FPLC纯化的mRNA疫苗，在小鼠、雪貂、非人灵长类动物以及首次人类中针对流感HA 10神经氨酸酶8（H10N8）和H7N9流感病毒的免疫原性[22]。用低剂量（0.4-10μg）编码流感病毒HA的LNP复合mRNA进行单次皮内或肌肉内免疫，在小鼠中引起针对同源流感病毒攻击的保护性免疫反应。在用一剂或两剂含有编码HA的LNP复合mRNA的50-400μg疫苗免疫后，雪貂和食蟹猴（cynomolgus monkeys）获得了类似的结果，证实了mRNA-LNP疫苗在大型动物（包括非人灵长类动物）中的的效力转化。

基于这些令人鼓舞的临床前数据，最近启动了两项I期临床试验，以评估核苷修饰的mRNA-LNP疫苗在人体中的免疫原性和安全性。编码H10N8 HA的mRNA疫苗目前正在进行临床试验（NCT03076385），并报告了23名接种疫苗者的中期结果[22]。参与者肌肉注射少量（100μg）疫苗，并在接种疫苗后43天测量免疫原性。通过血凝抑制和微中和抗体测定法测量，该疫苗在所有受试者中均具有免疫原性。有希望的是，抗体滴度高于预期的保护阈值，但略低于动物模型。与Alberer等人的研究类似[91]，大多数接种疫苗的受试者报告了轻度至中度的反应原性（注射部位疼痛，肌痛，头痛，疲劳和发冷），三名受试者报告了严重的注射部位反应或全身性普通感冒样反应。这种反应原性水平似乎与更传统的疫苗形式相似[113,114]。最后，Richner等人描述的寨卡病毒疫苗[85,112]也在I/II期联合试验（NCT03014089）中进入临床评估。未来应用核苷修饰的mRNA-LNP疫苗对抗更多样化的抗原的研究，将揭示该策略在多大程度上广泛适用于传染病疫苗。

mRNA癌症疫苗

对mRNA的癌症疫苗已有全面回顾[115-119]。下面重点介绍最新的进展和方向。癌症疫苗和其他免疫疗法有望替代现有治疗恶性肿瘤的方法。设计癌症疫苗，用来对抗在癌细胞中优先表达的肿瘤相关抗原（例如生长相关因子，或由于体细胞突变而对恶性细胞特有的抗原[120]）。这些新抗原（neoantigens）或其中的新表位（neoepitopes）已被部署为人类的mRNA疫苗靶标[121]（框2）。大多数癌症疫苗是治疗性的，而不是预防性的（prophylactic），寻求刺激细胞介导的反应（例如来自CTL的反应，这些反应能够清除或减轻肿瘤负担[122]）。二十多年前，第一批概念验证研究发表，不仅提出了RNA癌症疫苗的想法，而且还提供了这种方法可行性的证据[123,124]。从那时起，许多临床前和临床研究已经显示mRNA疫苗对抗癌症的可行性（见文后 表 3）。

1.树突状细胞mRNA癌症疫苗

由于树突状细胞（DC）是启动抗原特异性免疫反应的核心参与者，因此很自然就想到把它们用于癌症免疫治疗。Boczkowski及其同事在1996年首次报道，用mRNA电穿孔的DC可以引发针对肿瘤抗原的有效免疫反应的证据（参考文献124）。在这项研究中，用卵清蛋白（OVA）编码的mRNA或肿瘤衍生RNA脉冲的DC在小鼠表达OVA和其他黑色素瘤模型中引起肿瘤减少的免疫反应。已经以mRNA编码佐剂的形式鉴定出多种免疫调节蛋白，可以增加DC癌症疫苗的效力。几项研究表明，用编码共刺激分子（如CD83）、肿瘤坏死因子受体超家族成员4（TNFRSF4;也称为OX40）和4-1BB配体（4-1BBL）的mRNA对DC进行电穿孔导致DCs免疫刺激活性的大幅增加[125-128]。DC功能也可以通过使用mRNA编码的促炎细胞因子（如IL-12）或运输相关分子来调节[129-131]。如上所述，TriMix 是 mRNA 编码佐剂（CD70、CD40L 和组成活性 TLR4）的混合物，可与抗原编码的 mRNA 或 mRNA 结合使用电穿孔[132]。该配方通过增加DC活化并将CD4+ T细胞表型从T调节细胞转移到T辅助1（TH1）样细胞[132-136]，在多项临床前研究中证明是有效的。值得注意的是，使用装有编码黑色素瘤相关抗原的mRNA和TriMix佐剂的DC对III期或IV期黑色素瘤患者进行免疫接种，导致27%的治疗个体肿瘤消退[137]。现已使用DC疫苗进行了多项临床试验，针对各种癌症类型（例如转移性前列腺癌、转移性肺癌、肾细胞癌、脑癌、黑色素瘤、急性髓系白血病、胰腺癌等[138,139]）（在参考文献51，58中审查）。

一项新的研究，将DC的mRNA电穿孔与传统化疗药物或免疫检查点抑制剂相结合。在一项试验中，III 期或 IV 期黑色素瘤患者接受 ipilimumab（一种抗 CTL 抗原 4 （CTLA4） 的单克隆抗体）和装有编码黑色素瘤相关抗原的 mRNA 的 DC 和 TriMix 进行治疗。这种干预使一定比例的复发性或难治性黑色素瘤患者的肿瘤持久减少[140]。

2.mRNA癌症疫苗的直接注入

mRNA疫苗的给药途径和递送形式会极大地影响结果。已经开发了多种 mRNA 癌症疫苗形式，使用常见的递送途径（皮内、肌肉内、皮下或鼻内）和一些非常规的疫苗接种途径（结内、静脉内、脾内或肿瘤内）。

结内施用裸mRNA是一种非常规但有效的疫苗递送方式。将mRNA直接注入次级淋巴组织中，优势在于，在T细胞活化部位将抗原靶向递送至抗原呈递细胞，从而无需迁移DC。多项研究表明，结节内注射的裸mRNA可以被DC选择性地吸收，并可以引发有效的预防或治疗性抗肿瘤T细胞反应[62,66]；一项早期研究也证明了脾内分娩的类似发现[141]。在某些情况下，DC激活蛋白FMS相关酪氨酸激酶3配体（FLT3L）的共同给药显示可进一步改善对结内mRNA疫苗接种的免疫反应[142,143]。将TriMix佐剂掺入具有编码肿瘤相关抗原的mRNA的小鼠的结内注射中，可在多个肿瘤模型中产生有效的抗原特异性CTL反应和肿瘤控制[133]。最近的一项研究表明，用TriMix结内注射编码人瘤病毒（HPV）16的E7蛋白的mRNA增加了肿瘤浸润CD8+ T细胞的数量，并抑制了小鼠中表达E7的肿瘤模型的生长[67]。

由于临床前研究的成功，临床试验启动，在晚期黑色素瘤患者（NCT01684241）和肝细胞癌患者（EudraCT：2012-005572-34）中使用结节内注射编码肿瘤相关抗原的裸mRNA。在一项已发表的试验中，转移性黑色素瘤患者接受结节内给药的DC进行治疗，这些DC电穿孔了编码黑色素瘤相关抗原酪氨酸酶或gp100和TriMix的mRNA，可诱导有限的抗肿瘤反应[144]。

鼻内疫苗给药是一种无针、无创的递送方式，可使 DC 快速摄取抗原。鼻内递送的mRNA与Stemfect（Stemgent）LNP复合，在使用表达OVA的 E.G7-OVA T淋巴母细胞系的预防性和治疗性小鼠肿瘤模型中，可延迟肿瘤发作并增加存活率[145]。

肿瘤内mRNA疫苗接种，具有快速和特异性激活肿瘤驻留T细胞的优势。通常，这些疫苗不会引入编码肿瘤相关抗原的mRNA，目的是在免疫刺激分子原位激活肿瘤特异性免疫。一项早期研究表明，利用mRNA的内在免疫原性特性，编码非肿瘤相关基因（GLB1）的裸mRNA或鱼精蛋白稳定的mRNA会破坏肿瘤生长，对胶质母细胞瘤小鼠模型提供保护[146]。最近的一项研究表明，在表达OVA的淋巴瘤或肺癌小鼠模型中，编码基于干扰素-β（IFNβ）的工程细胞因子的mRNA与转化生长因子β（TGFβ）拮抗剂融合的mRNA的肿瘤内递送增加了CD8 + T细胞的细胞溶解能力，并适度延迟了肿瘤生长[147]。研究还表明，肿瘤内施用不编码肿瘤相关抗原的TriMix mRNA，会导致CD8α + DC和肿瘤特异性T细胞的活化，从而导致各种小鼠模型中肿瘤生长延迟[148]。

由于担心mRNA疫苗与血清蛋白聚集和细胞外mRNA快速降解，全身用药并不常见。因此，将mRNA配制成载体分子（carrier molecules）至关重要。如上所述，已经开发出许多递送配方来促进mRNA摄取，增加蛋白质翻译并保护mRNA免受RNases的侵害[10,11,79,80]。另一个重要问题是mRNA疫苗在全身递送后的生物分布（biodistribution）。某些基于阳离子LNP的络合剂主要通过静脉内输送到肝脏[21]，这可能不适合DC激活。最近出现了在全身递送后靶向mRNA疫苗DC的有效策略[59]。使用阳离子脂质和用mRNA配制的中性辅助脂质生成了mRNA-脂质复合物（mRNA-脂质体复合物）递送平台，脂质与mRNA的比例以及颗粒的净电荷对疫苗的生物分布具有深远的影响。虽然带正电荷的脂质颗粒主要针对肺部，但带负电荷的颗粒针对次级淋巴组织和骨髓中的DC。带负电荷的颗粒诱导了针对肿瘤特异性抗原的有效免疫反应，这些抗原与各种小鼠模型中令人印象深刻的肿瘤大量减少有关[59]。由于在小鼠或非人灵长类动物中未观察到毒性作用，因此已启动使用这种方法治疗晚期黑色素瘤或三阴性乳腺癌患者的临床试验（NCT02410733和NCT02316457）。

皮肤中存在多种抗原呈递细胞[149]，因此皮肤是疫苗接种期间免疫原递送的理想部位（图3）。皮内递送途径已广泛用于mRNA癌症疫苗。一项早期的开创性研究表明，皮内施用肿瘤RNA可延缓纤维肉瘤小鼠模型中的总体肿瘤生长[65]。在基于鱼精蛋白的RNActive平台中皮内注射编码肿瘤抗原的mRNA在各种癌症小鼠模型中证明是有效的[36]，并且在多种预防和治疗临床环境中（表3）。一项研究表明，编码存活蛋白和各种黑色素瘤肿瘤抗原的mRNA，导致黑色素瘤患者亚群中抗原特异性T细胞的数量增加[150]。在患有去势抵抗性（castration-resistant）前列腺癌的人类中，表达多种前列腺癌相关蛋白的RNActive疫苗，在大多数接受者中引起抗原特异性T细胞反应[151]。基于脂质的载体，也有助于皮内递送的mRNA癌症疫苗的功效。在DOTAP和/或DOPE脂质体中递送OVA编码的mRNA导致抗原特异性CTL活性并抑制小鼠中表达OVA的肿瘤的生长[152]。在同一项研究中，编码粒细胞-巨噬细胞集落刺激因子（GM-CSF）的mRNA的共同给药改善了OVA特异性细胞溶解反应。另一份报告显示，皮下递送编码两种黑色素瘤相关抗原的LNP配制的mRNA可延缓小鼠肿瘤生长，LNP中脂多糖（LPS）的共同递送可增加CTL和抗肿瘤活性[153]。总体来说，mRNA癌症疫苗已被证明在人类中具有免疫原性，但根据基础免疫学研究，可能需要改进疫苗的接种方法，以获得更大的临床益处。

在一些临床前研究中，mRNA疫苗接种与传统化疗、放疗和免疫检查点抑制剂等辅助疗法相结合，改善了疫苗接种的结果[154,155]。例如，顺铂（cisplatin）治疗显著提高了编码HPV16 E7癌蛋白和TriMix的mRNA免疫的治疗效果，引起小鼠模型中磁性生殖道肿瘤的完全排斥反应[67]。值得注意的是，也有人提出，用针对程序性细胞死亡蛋白1（PD1）的抗体治疗，提高了基于新表位mRNA的疫苗对人类转移性黑色素瘤的疗效，但需要更多的数据来验证这一假说[68]。

1. The table summarizes the clinical trials registered at ClinicalTrials.gov as of 5 May 2017. Ag, antigen; AML, acute myeloid leukaemia; CD40L, CD40 ligand; CML, chronic myeloid leukaemia; CMV, cytomegalovirus; DC, dendritic cell; EP, electroporated; GM-CSF, granulocyte–macrophage colony-stimulating factor; i.d., intradermal; ing., inguinal injection; i.nod., intranodal injection; i.v., intravenous; NA, not available; neo-Ag, personalized neoantigen; s.c., subcutaneous; TAA, tumour-associated antigen.

2. *Developed by CureVac AG.

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