摘要
呼吸道黏膜免疫系统是机体抵御感染的第一道防线,自2019年新型冠状病毒肺炎(corona virus disease 2019,COVID-19)发生以来,鼻腔黏膜免疫疫苗因具有诱导细胞、体液及黏膜三重免疫应答能力,已成为研究的热点。本文聚焦于新型冠状病毒,了解其结构及致病机制,并对鼻腔黏膜免疫机制进行简要介绍,概述鼻腔黏膜免疫疫苗种类及临床研究进展,以期为新型疫苗的研发提供理论参考,寻求预防治疗COVID-19的最佳方法与策略。
2019年,由高传播性和致病致死性的新型冠状病毒严重急性呼吸综合征冠状病毒2型(severe acute respiratory syndrome coronavirus 2,SARS-CoV-2)引起的COVID-19疫情迅速蔓延全球,给人类的生命健康安全带来巨大威胁。截至目前,控制疫情和预防死亡的最佳策略仍然是接种疫苗,设计具有预防和治疗作用的新型疫苗迫在眉睫。
疫苗主要给药途径为肌肉注射、皮下注射或皮内注射,但注射人员须经专门培训,且注射有产生疼痛和急性感染的风险,患者依从性差,同时在应对SARS-CoV-2等呼吸道病毒时不足以防止病毒传播。因此,探索疫苗递送的非侵入性给药途径具有必要性。黏膜免疫由于其独特的优势成为疫苗研究的热点,其中鼻腔免疫接种作为全身疫苗接种的替代方法,与注射给药相比,更为安全有效,可有效诱导免疫球蛋白A(immunoglobulin A,IgA),激发鼻腔和上呼吸道中的常驻记忆B和T细胞,进而产生细胞免疫、体液免疫及黏膜免疫三重免疫应答,阻止病毒的复制与传
SARS-CoV-2是一种单链正链RNA(+ssRNA)病毒,具有单一线性RNA片段,属于冠状病毒家族。如

Figure 1 Structure of SARS-CoV-2 virus
SARS-CoV-2冠状病毒进入宿主细胞由S蛋白介导。S蛋白在病毒表面形成同型三聚体,负责病毒与宿主细胞膜的附着和融合。S蛋白包含S1和S2两个结构域,其中S1结构域含有的受体结合域(receptor binding domain,RBD)负责与细胞表面受体血管紧张素转换酶2(angiotensin-converting enzyme 2,ACE2)结合,S2结构域负责病毒膜和细胞膜融合,从而允许病毒进入细胞(

Figure 2 Mechanism of SARS-CoV-2 entering human cellsACE2: Angiotensin-converting enzyme 2
新冠病毒的主要传播途径是呼吸道传播和接触传播。接触传播主要来源于感染者释放的病毒颗粒/飞沫沉降到物体表面,健康者手接触后沾染病毒,在不经意间触碰到鼻腔、口腔或者眼睛结膜等黏膜时,病毒就容易入侵人体,导致感染;呼吸道传播是病毒携带者通过打喷嚏、咳嗽或交谈,以飞沫或气溶胶的形式在空气中释放病毒微粒,以空气为介质进行传播,将病毒传染给健康者。研究证明,鼻上皮中ACE2的浓度最高,可以推断病毒颗粒的复制主要发生在鼻腔黏
黏膜相关淋巴组织(mucosal-associated lymphoid tissue,MALT)亦称黏膜免疫系统(mucosal immune system,MIS),是指广泛分布于呼吸道、胃肠道、泌尿生殖道黏膜下及一些外分泌腺体处的淋巴组织,是执行局部特异性免疫功能的主要场所。大部分病毒和细菌感染始于黏膜表面,MALT构成了机体的第一道防线。黏膜表面与外界抗原直接接触,在外来病原微生物或其他外来抗原侵入机体组织之前将其消灭,使其无法损伤机体,在抵抗感染方面发挥极其重要的作
NALT是上呼吸道主要的免疫防御屏障,是鼻腔接种疫苗后黏膜免疫反应的主要诱导部位,鼻腔接种疫苗后NALT的免疫反应机制见

Figure 3 Mechanism of NALT immune response after nasal vaccinatio
NALT能够同时产生细胞、体液和黏膜三重免疫应答,而其他黏膜组织(如,口腔黏膜和阴道黏膜)并不具有这一特性。由于鼻黏膜给药独特的免疫优势,使得鼻腔免疫疫苗成为当前研究的热点。细胞免疫反应通过抗体依赖细胞介导的细胞毒反应和直接细胞毒性T 细胞来实现,能够直接杀死特殊的感染细胞,因而对细菌、病毒和寄生虫等的清除至关重要。黏膜部位的体液免疫反应通过使活化的B 细胞分泌二聚体或多聚体形式的IgA来实现,能够抵抗酶的降解,发挥黏膜免疫作用。SIgA是抵御病原体入侵的第一道屏障,它可以结合并中和黏液中的病原体或毒素,阻止病毒与黏膜表面直接接触,进而将病毒清除。因此诱导有效的IgA应答是黏膜免疫疫苗成功接种的先决条件,是实现黏膜免疫的关
研究表明,鼻纤毛细胞是SARS-CoV-2最初的感染和复制部位,SARS-CoV-2一旦到达肺部,会导致严重肺炎并伴有其他疾病的发
DNA疫苗的原理是通过一定的途径使编码免疫原性抗原的重组质粒载体进入人体内表达抗原,诱导人体产生免疫应答。因与传统疫苗相比,DNA疫苗具有研发和生产周期短、稳定性高,能诱导更强大的细胞毒性T细胞反应,且没有严重的不良反应等优
DNA疫苗是一种针对特定蛋白的抗体疫苗,已被证明擅长触发细胞免疫反应,且更快速、更容易放大生产,因此鼻腔黏膜免疫DNA疫苗的开发显示出较好的应用前景。研究人员采用基因转移技术开发了一种新型鼻腔喷雾COVID-19疫苗,该疫苗以腺病毒为载体递送克隆的DNA链,能使鼻咽细胞产生病毒蛋白,进而对疫苗产生反应,并且在动物研究中表现良好,人体临床试验也将逐步开展,有望获得上市授
RNA疫苗因其只需进入细胞质而不需进入细胞核内即能发挥作用,所以安全性相对较高,这也促使RNA疫苗成为当前新型核酸疫苗研究的热
病毒载体疫苗,即使用经过修改的无害的病毒为载体将抗原(如SARS-CoV-2 S蛋白)的遗传密码(DNA或RNA)传递到人体细胞,模拟自然病毒感染,以产生所需的免疫反
研究人员利用复制能力不强、无致病性的人副流感病毒2型 (human parainfluenza virus2,HPIV2)载体BC-PIV研制SARS-CoV-2鼻腔疫苗。研究表明,表达前预融合稳定S蛋白的BC-PIV在小鼠鼻内接种疫苗后可诱导产生抗SARS-CoV-2 的血清中和IgG和鼻黏膜IgA抗体,从而预防呼吸道的病毒感染,且无明显的不良反
基因重组亚单位疫苗仅包含源自致病性细菌或病毒的某些成分,这些成分是能够引起机体免疫反应的主要物质,由于其不含完整病原体,因此安全性好,在临床研究中显示出较好的潜力。
干扰素基因刺激物 (stimulator of interferon genes,STING)能够感知胞浆核酸,并激活TANK结合激酶-1(TANK binding kinase 1,TBK1)/干扰素调节因子-3(interferon regulatory factor 3,IRF-3)信号通路,诱导促炎细胞因子和干扰素-β(interferon-β,IFN-β)的表
如前所述,自SARS-CoV-2基因组序列发布以来,针对COVID-19的一系列疫苗平台被开发利用,多种候选疫苗处于临床评估阶段,主要以SARS-CoV-2 S蛋白及RBD为靶
Phase | Vaccine | Type | Developer/manufacturer | Enrollment | Clinical trial No. | Status |
---|---|---|---|---|---|---|
I | Ad5-nCoV | Ad-vectored vaccine | CanSino/Beijng Institute of Biotechnology | 149 | NCT04552366 | Active,not recruiting |
ChAdOx1nCOV-19 | Ad-vectored vaccine | University of Oxford | 42 | NCT04816019 | Complete | |
BBV154 | Ad-vectored vaccine | Bharat Biotech Intemational Limited | 175 | NCT04751682 | Complete | |
SC-Ad6-1 . | Ad-vectored vaccine | Tetherex Pharmaceuticals Corporation | 130 | NCT04839042 | Recruiting | |
AdCOVID | Ad-vectored vaccine | Altimmune, Inc. | 92 | NCT04679909 | Active,not recruiting | |
NDV-HXP-S | NDV-vectored vaccine | Sean Liu,Icahn School of Medicine at Mount Sinai | 35 | NCT05181709 | Recruiting | |
AVX/COVID-12-HEXAPRO | NDV-vectored vaccine | LaboratorioAvi-Mex. SA de CV. | 91 | NCT04871737 | Complete | |
DelNS1-nCoV-RBD LAIV | LAIV-vectored vaccine | The University of Hong Kong | 115 | NCT04809389 | Active,not recruiting | |
DelNS1-2019-nCoV-RBD-OPT1 | LAIV-vectored vaccine | University of Hong Kong. Xia men University and Being Wantai Biological Pharmacy | 60 | ChiCTR000037782 | Complete | |
CVXGA1 | PIV5-vectored vaccine | CyanVac LLC | 80 | NCT04954287 | Recruiting | |
MV-014- 212 | RSV-vectored vaccine | Meissa Vaccines,Inc. | 130 | NCT04798001 | Recruiting | |
Razi CoV Pars | Protein subunit vaccine | Razi Vaccine and Serum Research Institute | 153 | IRCT20201214049709N1 | Complete | |
Gam-COVID-Vac | two-component combined Ad-vector vaccine | Gamaleya Research Institute of Epidemiology and Microbiology,Health Ministry of the Russian Federation | 400 | NCT05248373 | Not yet recruiting | |
COVI-VAC | Live attenuated vaccine | Codagenix. Inc. | 48 | NCT04619628 | Complete | |
GLS-5310 | DNA vaccine | GeneOne Life Science,Inc. | 69 | NCT05182567 | Active,not recruiting | |
Avacc 10 | OMV vaccine | Intravacc B.V. | 36 | NCT05604690 | Not yet recruiting | |
II | BBV154 | Ad-vectored vaccine | Bharat Biotech Intemational Limited | 608 | NCT05639998 | Complete |
AVX/COVID-12 | NDV-vectored vaccine | LaboratorioAvi-Mex. SA de CV. | 158 | NCT05205746 | Active,not recruiting | |
Razi CoV Pars | Protein subunit vaccine | Razi Vaccine and Serum Research Institute | 500 | IRCT20201214049709N2 | Complete | |
DelNS1-2019-nCoV-RBD-OPT1 | LAIV-vectored vaccine | University of Hong Kong. Xia men University and Being Wantai Biological Pharmacy | 720 | ChiCTR2000039715 | Complete | |
III | BBV154 | Ad-vectored vaccine | Bharat Biotech Intemational Limited | 3 160 | NCT05522335 | Active,not recruiting |
Razi CoV Pars | Protein subunit vaccine | Razi Vaccine and Serum Research Institute | 41 128 | IRC720210206050259N3 | Complete | |
DelNS1-2019-nCoV-RBD-OPT1 | Influenza Virus Vector | N/A | 20 000 | ChiCTR2100051391 | Complete |
Ad: Adenovirus; NDV: Newcastle disease virus; LAIV: live attenuated influenza virus; PIV: parainfluenza virus; RSV: respiratory syncytial virus; OMV: outermembrane vesicles; N/A: Not available. Data from https://clinicaltrials.gov/,https://covid-19.cochrane.org/andhttps://trialsearch.who.int/Default.aspx
COVID-19鼻腔疫苗主要以病毒载体疫苗开发为主,我国康希诺生物研发团队研发了全球首款可吸入用新冠疫苗克威
当前全世界范围内COVID-19疫情仍在肆虐,接种疫苗是防止疫情迅速大范围传播最有效的方法。免疫途径的选择对于诱导有效免疫保护至关重要,黏膜表面是病原体进入并感染机体的主要部位。SARS-CoV-2病毒变异快,感染路径由上呼吸道传播转为下呼吸道传播,甚至会同时攻击上呼吸道和下呼吸道,因此建立呼吸道黏膜免疫屏障是对抗COVID-19的有效策略。鼻腔黏膜免疫疫苗作为一种新型非侵入式疫苗给药系统,具有使用方便、安全有效、患者顺应性好等特点,并能提高免疫治疗效果,诱导细胞、体液和黏膜免疫应答,实现三重免疫,在新冠免疫疫苗的未来发展方向上,鼻腔疫苗接种或将成为预防COVID-19等呼吸道传染病的有效途径。
与此同时,鼻腔黏膜免疫仍存在一些问题有待改进,如抗原的稳定性、有效摄取及免疫佐剂的安全性,同时由于SARS-CoV-2 病毒变异快,不断出现逃逸株,难以形成针对变异株的群体免疫,迫切需要研发新型抗新冠疫苗。采用广谱的免疫原设计高度活化中和抗体,与T细胞应答协同抗击病毒并有效清除病毒,构建群体有效免疫保护;采用鼻腔黏膜接种或黏膜与肌肉序贯接种,以实现黏膜持久应答,加强免疫反应,持续防御病毒,防止病毒持续变异。随着对鼻腔黏膜免疫机制及COVID-19鼻腔黏膜免疫疫苗研究的深入,将使鼻腔黏膜免疫在COVID-19疫情中发挥更强有力的作用,对传染病的防控具有重要意义。
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