【作者】 陈利；

【导师】 魏霞蔚；

【作者基本信息】 四川大学 ， 药学， 2023， 硕士

【摘要】 目的:随着严重急性呼吸综合征冠状病毒2（severe acute respiratory syndrome coronavirus 2,SARS-Co V-2）感染所致的新型冠状病毒肺炎（coronavirus disease 2019,COVID-19）在全球蔓延,接种有效、安全的疫苗是阻止病毒传播和控制大流行的最佳策略之一。完成基础免疫后,新冠疫苗预防COVID-19的有效性随着时间的推移而下降,在新的变异株不断引发新一轮疫情爆发的背景下,接种疫苗加强针已经成为必然趋势。根据不同的研发技术路线,新冠疫苗主要包括灭活病毒疫苗、蛋白亚单位疫苗、腺病毒载体疫苗、核酸疫苗等4种疫苗。序贯接种不同技术平台的疫苗可以形成优势互补,以提高免疫反应的强度、种类,并增加中和抗体的广度和持久性。然而,通过肌肉注射接种的新冠疫苗主要引起全身性免疫应答,难以诱导出针对新冠病毒的黏膜免疫反应。采用肌肉免疫与滴鼻免疫相结合的方式,则可以诱导产生细胞免疫、体液免疫及黏膜免疫三重免疫应答,为机体提供更全面的保护。本研究旨在肌肉注射mRNA疫苗作为基础免疫,后采用滴鼻免疫RBD-HR蛋白疫苗作为序贯加强策略,探究新冠蛋白亚单位疫苗序贯免疫能否引发广泛的免疫反应,为预防新冠病毒的感染和传播提供思路和方法。方法:对NIH小鼠肌肉注射2剂mRNA疫苗,后采用滴鼻的方式免疫2剂RBD-HR蛋白疫苗,每次免疫间隔21天。末次免疫后14天,收集免疫小鼠的血清。首先,对血清进行一系列梯度稀释后,通过酶联免疫吸附试验（enzyme linked immunosorbent assay,ELISA）检测免疫小鼠产生的特异性结合抗体滴度,包括免疫血清的IgG、IgG1、IgG2a、IgG2b、IgG2c、IgG3、IgA,鼻腔灌洗液（nasal lavage fluid,NLF）的分泌型IgA（secretory IgA,SIgA）,支气管肺泡灌洗液（bronchoalveolar lavage fluid,BALF）的SIgA。其次,为评估免疫血清对RBD-ACE2结合的阻断能力,对免疫血清进行1:90倍稀释后,进行阻断实验。采用流式细胞仪检测RBD-Fc蛋白（包括WT、B.1.617.2、BA.1）与ACE2-293T细胞的结合情况,并计算血清的抑制率。随后,为评估免疫血清对新冠假病毒的中和能力,选择6种新冠假病毒进行假病毒中和试验,包括野生型、Delta（B.1.617.2）、Omicron（BA.1）、Omicron（BA.2）、Omicron（BA.3）、Omicron（BA.4/5）。通过荧光显微镜观察免疫血清对野生型新冠假病毒的中和现象,并使用流式细胞仪和生物发光酶标仪量化免疫血清对不同突变的新冠假病毒的中和能力,计算50%假病毒中和滴度（50%pseudovirus neutralization titer,p VNT50）。同样地,末次免疫后14天,通过流式细胞术评估RBD-HR滴鼻序贯免疫引发的黏膜免疫反应,包括支气管肺泡灌洗液和肺组织中CD103⁺树突状细胞（dendritic cells,DC）、记忆性T细胞、组织常驻记忆T细胞(tissue resident memory T cells,T_RM)。同时,将免疫小鼠的肺组织细胞与新冠病毒相关肽库共培养12 h后,收集细胞并进行胞内染色,通过流式细胞术检测TNF-α、IL-2的分泌情况。最后,检测免疫小鼠纵隔淋巴结、腹股沟淋巴结、脾脏等三个主要免疫器官中滤泡辅助T细胞（T follicular helper cells,Tfh）和生发中心B细胞（germinal center B cells,GC B）的产生,评价RBD-HR滴鼻序贯免疫诱导的系统性免疫反应。免疫期间,观察小鼠饮水、采食等行为状态,并记录小鼠体重,免疫程序结束后,收集小鼠主要器官心、肝、脾、肺、肾,进行H&E染色,评估mRNA疫苗联合RBD-HR疫苗进行序贯免疫的安全性。结果:肌肉免疫mRNA疫苗（0.1μg/只或1μg/只）后,滴鼻免疫RBD-HR疫苗,免疫小鼠血清中产生的特异性IgG、IgG1、IgG2a、IgG2b、IgG2c、IgG3、IgA抗体水平均显著高于mRNA疫苗组,且IgG1抗体滴度远高于IgG2a。1:90倍稀释免疫血清,滴鼻序贯免疫组血清对不同突变株的RBD-Fc（WT、Delta、Omicron）与血管紧张素转换酶2（angiotensin converting enzyme 2,ACE2）的结合有明显的抑制作用,且高剂量mRNA滴鼻序贯免疫RBD-HR疫苗的抑制作用更显著。随后我们发现滴鼻序贯免疫RBD-HR疫苗后,免疫血清对野生型和不同突变的新冠假病毒（WT、B.1.617.2、BA.1、BA.2、BA.3、BA.4）的50%假病毒中和滴度比mRNA疫苗组高,且具有统计学差异。在诱导产生的黏膜免疫中发现:与mRNA疫苗组相比,滴鼻序贯免疫可诱导鼻腔灌洗液和支气管肺泡灌洗液中产生更高滴度的特异性SIgA抗体;诱导支气管肺泡灌洗液和肺组织产生高水平CD103⁺树突状细胞并促进其活化。此外,滴鼻序贯免疫可以促进支气管肺泡灌洗液和肺组织中T细胞及记忆性CD4⁺T细胞、记忆性CD8⁺T细胞、组织常驻记忆CD4⁺T细胞、组织常驻记忆CD8⁺T细胞数量增加。使用新冠相关肽库刺激肺组织细胞,可诱导记忆性CD4⁺和CD8⁺T细胞分泌Th1型细胞因子。明确mRNA疫苗联合RBD-HR疫苗序贯免疫能诱导更强的体液免疫和黏膜免疫后,进一步发现单独mRNA疫苗可以诱导主要免疫器官纵隔淋巴结、腹股沟淋巴结、脾脏滤泡辅助T细胞、生发中心B细胞生成,滴鼻序贯免疫则可显著增加滤泡辅助T细胞、生发中心B细胞的数量。免疫期间,所有组别免疫小鼠的状态良好、体重变化稳定,主要器官的组织病理切片未见明显病理变化。结论:肌肉免疫mRNA疫苗作为基础免疫,滴鼻RBD-HR疫苗作为加强免疫的序贯策略可以同时诱导机体产生广泛高效的体液免疫、黏膜免疫和细胞免疫,达到三重免疫保护作用。与单独免疫mRNA疫苗相比,滴鼻序贯免疫可产生高滴度的特异性IgG及其亚型、血清型IgA、SIgA,且可以增强免疫血清对RBD-ACE2的阻断能力、增强对不同突变型新冠假病毒的中和能力。激活体液免疫的同时,滴鼻序贯免疫在肺部促进CD103⁺树突状细胞、记忆性T细胞的产生及分化,并诱导生发中心的形成,激发系统性免疫反应,具有更强的免疫保护效果。综上所述,以RBD-HR蛋白疫苗作为滴鼻的序贯策略可安全高效地诱导机体产生广泛的免疫反应,为优化新冠疫苗有效性和持久性提供潜在策略。更多还原

【Abstract】 Objective:As severe acute respiratory syndrome coronavirus 2（SARS-Co V-2）continues to spread globally,vaccination with effective and safe vaccines has become one of the best strategies to prevent virus transmission and control the pandemic.However,the efficacy of SARS-Co V-2 vaccines in preventing COVID-19 decreases over time after completion of the primary immunization,and with the emergence of new variant strains causing new waves of outbreaks,booster shots have become a necessary trend.Based on different development technologies,SARS-Co V-2 vaccines mainly include inactivated vaccines,subunit protein vaccines,adenovirus vector vaccines,and nucleic acid vaccines.Heterologous vaccination with different technology platforms can form complementary advantages to enhance the intensity and diversity of immune responses,and increase the breadth and persistence of neutralizing antibodies.However,SARS-Co V-2 vaccines administered by intramuscular injection mainly induce systemic immune responses and are difficult to induce mucosal immune responses against SARS-Co V-2.Using a combination of intramuscular and intranasal immunization can induce triple immune responses including cellular,humoral,and mucosal immunity,providing more comprehensive protection for the body.This study aims to investigate whether intramuscular immunization with mRNA vaccine as the primary immunization,followed by intranasal immunization with RBD-HR protein vaccine as the booster strategy,can induce broad immune responses to SARS-Co V-2 subunit protein vaccines,and provide ideas and methods for preventing SARS-Co V-2 infection and transmission.Methods:NIH mice were intramuscularly injected with 2 doses of mRNA vaccine,followed by 2 doses of RBD-HR protein vaccine administered via nasal drops,with a 21-day interval between each immunization.14 days after the final immunization,the serum was collected from the immunized mice.Firstly,a series of gradient dilutions were performed on the serum,and the titers of specific binding antibodies produced by the immunized mice were detected by enzyme-linked immunosorbent assay（ELISA）,including IgG,IgG1,IgG2a,IgG2b,IgG2c,IgG3,and IgA in the serum,and secretory IgA（SIgA）in nasal lavage fluid（NLF）and bronchoalveolar lavage fluid（BALF）.Secondly,to evaluate the blocking ability of the serum against RBD-ACE2 binding,the serum was diluted 1:90 and RBD-ACE2 blocking experiment was performed.The binding of RBD-Fc protein（including WT,B.1.617.2,and BA.1）to ACE2-293T cells was detected using a flow cytometer,and the serum inhibition rate was calculated.Then,to evaluate the neutralizing ability of the serum against SARS-Co V-2 pseudovirus,six types of SARS-Co V-2 pseudovirus were selected for pseudovirus neutralization assays,including wild-type（WT）,Delta（B.1.617.2）,Omicron（BA.1）,Omicron（BA.2）,Omicron（BA.3）,and Omicron（BA.4/5）.The phenomenon of the serum against WT SARS-Co V-2pseudovirus was observed using fluorescence microscopy,and the neutralizing ability of the serum against SARS-Co V-2 pseudoviruses with different mutations was quantified using a flow cytometer and a bioluminescent enzyme-linked immunosorbent assay,and the 50%pseudovirus neutralization titer（p VNT50）was calculated.Similarly,14days after the final immunization,mucosal immune responses were detected by flow cytometry,including CD103⁺dendritic cells（DC）,memory T cells,and tissue resident memory T cells(T_RM)in BALF and lung tissue.Additionally,after co-culturing lung tissue cells from immunized mice with SARS-Co V-2 peptide pool for 12 h,cells were collected and subjected to intracellular staining to detect the secretion of TNF-αand IL-2 by flow cytometry.Finally,the formation of Tfh and GC B in the main immune organs,including mediastinal lymph nodes,inguinal lymph nodes,and spleen,was detected to evaluate the systemic immune response induced by intranasal boost with RBD-HR protein vaccine.During the immunization period,the behavior of the mice,including drinking and feeding,was observed,and the body weight of the mice was investigated.After the immunization procedure was completed,the main organs of the mice,including the heart,liver,spleen,lungs,and kidneys,were collected and subjected to H&E staining to evaluate the safety of the mRNA vaccine and RBD-HR vaccine.Results:After immunization with mRNA vaccine（0.1μg/dose or 1μg/dose）,intranasal immunization with RBD-HR vaccine resulted in significantly higher levels of IgG,IgG1,IgG2a,IgG2b,IgG2c,IgG3,and IgA antibodies in mouse serum compared to the mRNA vaccine group,and the IgG1antibody titer is much higher than that of IgG2a.After diluting the immune serum 1:90,the serum from the mRNA+RBD-HR group showed significant inhibitory effects on the binding of RBD-Fc（WT,Delta,Omicron）to（angiotensin converting enzyme 2,ACE2）receptor,and the inhibitory effect of the high-dose mRNA+RBD-HR group was more significant.Subsequently,we found that after intranasal boost with RBD-HR vaccine,the serum had a higher p VNT50 against WT and different mutant SARS-Co V-2 pseudoviruses（WT,B.1.617.2,BA.1,BA.2,BA.3,BA.4）than the mRNA vaccine,and there was a statistically significant difference.In terms of vaccine-induced mucosal immunity,we found that compared with the mRNA vaccine group,intranasal boost with RBD-HR vaccine could induce higher titers of specific SIgA in NLF and BALF,induce high levels of CD103⁺DC in BALF and lung tissue and promote their activation.In addition,intranasal boost with RBD-HR vaccine can significantly increase the number of T cells and memory CD4⁺T cells,memory CD8⁺T cells,CD4⁺T_RM,and CD8⁺T_RM in BALF and lung tissue.Stimulating lung tissue cells with SARS-Co V-2 peptide pool can induce memory CD4⁺and CD8⁺T cells to secrete Th1-type cytokines.After clarifying that intranasal boost with RBD-HR vaccine can induce stronger humoral and mucosal immunity,it was further found that the mRNA vaccine alone can induce partial formation of Tfh and GC B in the main immune organs such as the mediastinal lymph nodes,inguinal lymph nodes,and spleen,while intranasal boost with RBD-HR vaccine can significantly increase the number of Tfh and GC B,enhancing systemic immune responses.During the vaccination period,all immunized mice were in good condition with stable weight changes,and no significant pathological changes were observed in the histopathological sections of the main organs.Conclusion:Intramuscular immunization with mRNA vaccines followed by intranasal immunization with RBD-HR vaccines elicited a potent immune response,including humoral,mucosal,and cellular immune response.Compared to mRNA vaccination alone,intranasal boost with RBD-HR vaccine generated high titers of specific IgG,IgG subtypes,serum IgA,and SIgA,enhanced the ability of the serum to block RBD-ACE2 binding,and neutralized different variants of SARS-Co V-2 pseudoviruses.While activating the humoral immune response,intranasal boost with RBD-HR vaccine not only promoted the generation and differentiation of CD103⁺DC and memory T cells in the lungs,but also induced GC formation,thus stimulating systemic immunity.In summary,the utilization of RBD-HR protein vaccine as a heterologously intranasal immunization strategy was shown to be safe and effective in eliciting comprehensive immune response,which can potentially optimize the efficacy and durability of SARS-CoV-2 vaccines.更多还原