Objective:To investigate the effects of poly(A) tails with different lengths on mRNA expression in vitro and the passage stability of transcription template with poly (A) tail in Escherichia coli ( E. coli). Methods:Plasmids with poly(A) tails of 38, 60, 103, 125 and 126 (60 nt+ 6 nt spacer+ 60 nt) nt were designed and constructed. Then the plasmids were linearized by single enzyme digestion and used as transcription template for preparing enhanced green fluorescent protein (EGFP)-mRNA. EGFP-mRNA containing poly(A) tails of different lengths were transfected into 293T cells and the expression of EGFP was detected by flow cytometry. As to stability test, the template plasmids with poly (A) tail of 125 and 126 nt were transformed into E. coli TransStbl3 and Top10 competent cells. Seven clones were selected for culture and plasmid extraction, and then the plasmids were digested by restriction enzyme and detected by capillary electrophoresis. For passage stability, three correctly sequenced clones of each group were selected for continuous passage at 37℃, and the plasmids were extracted and digested every two generations for capillary electrophoresis. At the same time, the correctly sequenced clones of 125 nt group were also passaged at 30℃, and the plasmids were also extracted and digested every two generations for capillary electrophoresis. Results:The transcription templates with poly(A) tail of different lengths were successfully constructed. Flow cytometry showed that the fluorescence expression of the template plasmids with poly (A) tail of 103 and 125 nt were significantly higher than that of 38 and 60 nt. The fluorescence expression of the plasmid with poly (A) tail of 126 nt was significantly higher than that of all other groups. The percentages of stable sequences of the template plasmid with poly(A) tail of 125 nt in TransStbl3 and Top10 competent cells were 76% and 91%, respectively. The results of continuous passage showed that poly(A) tail of 125 nt could be stable to the 4th generation at 37℃ in both TransStbl3 and Top10 competent cells, and stable to the 16th and 10th generations at 30℃. The percentages of stable sequences of the template plasmid with poly(A) tail of 126 nt in TransStbl3 and Top10 competent cells were 95% and 48%, respectively. The results of continuous passage showed that poly(A) tail of 126 nt could be stable to the 12th generation at 37℃ in both TransStbl3 and Top10 competent cells.Conclusions:The length and composition of poly(A) tail in mRNA affected the expression of target protein. Adding a spacer with a length of 6 nt to poly(A) tail and low temperature culture were both helpful to improve the stability of the template plasmid, which provided a reference for the design and preparation of in vitro transcription template of mRNA vaccine.

The immunity of patients who recover from coronavirus disease 2019 (COVID-19) could be long lasting but persist at a lower level. Thus, recovered patients still need to be vaccinated to prevent reinfection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or its mutated variants. Here, we report that the inactivated COVID-19 vaccine can stimulate immunity in recovered patients to maintain high levels of anti-receptor-binding domain (RBD) and anti-nucleocapsid protein (NP) antibody titers within 9 months, and high neutralizing activity against the prototype, Delta, and Omicron strains was observed. Nevertheless, the antibody response decreased over time, and the Omicron variant exhibited more pronounced resistance to neutralization than the prototype and Delta strains. Moreover, the intensity of the SARS-CoV-2-specific CD4+ T cell response was also increased in recovered patients who received COVID-19 vaccines. Overall, the repeated antigen exposure provided by inactivated COVID-19 vaccination greatly boosted both the potency and breadth of the humoral and cellular immune responses against SARS-CoV-2, effectively protecting recovered individuals from reinfection by circulating SARS-CoV-2 and its variants.

目的 建立用于检测流感疫苗神经氨酸酶(neuraminidase,NA)活性的荧光底物法,并进行验证.方法 以唾液酸类似物4-MUNANA作为酶底物,通过测定NA催化底物分解后产物的荧光强度来评价NA活性.选取基于MDCK细胞制备的四价流感疫苗各单价原液(H1N1、H3N2、BV和BY)作为样品,优化荧光底物法的底物酶浓度(10、20、30、40、50 μmol/L)、作用时间(每 2 min 检测 1 次,至 90 min)、缓冲液 pH(4.5、5.5、6.5、7.5、8.5 和 9.5)及反应温度(4、25、37和45℃).以已知活性的重组NA蛋白作为参考品,验证方法的线性范围、准确度及精密度.用优化的方法检测流感疫苗生产过程中的样品(基于MDCK细胞制备的H1N1、H3N2、BV、BY相应的病毒收获液、纯化液和原液)及以MDCK细胞基质和鸡胚基质制备的四价流感裂解疫苗成品各1批.结果 最佳反应条件为:酶底物浓度20μmol/L,反应时间40 min,pH 7.5,反应温度37℃.重组NA蛋白参考品浓度在62.5～2 000 U/L范围内与荧光强度呈良好的线性关系,R2＞0.99;1 600、800、400 U/L 3个浓度参考品的回收率分别为92.60％～106.96％、85.54％～113.11％、87.00％～114.54％,于 2 个时间点共 12 次检测结果的 CV 分别为 6.40％、8.25％、6.92％.H1N1、H3N2、BV 和 BY 4种亚型的流感疫苗半成品在不同阶段NA活性逐渐降低;细胞基质及鸡胚基质四价流感裂解疫苗成品的NA活性分别为61 585.82和69 583.51 U/L.结论 成功建立并优化了一种NA活性的荧光底物检测方法,该方法具有良好的准确度和精密度,可应用于流感疫苗生产过程及成品中NA活性的检测.

目的 分析甲型H5N1流感病毒(简称H5N1)神经氨酸酶(neuraminidase,NA)生物信息学特征,原核表达获得H5N1 NA重组蛋白,并在动物模型中评价其免疫原性.方法 利用生物信息学软件分析H5N1 NA的理化性质、跨膜区、信号肽以及二级结构和高级结构.将H5N1 NA的编码区进行密码子优化后基因合成,将合成目的基因片段与pET-22b(+)连接,构建重组原核表达质粒pET-22b(+)-H5N1 NA,将重组质粒转化到BL21(DE3)感受态细胞并进行诱导表达;超声破碎菌体,使用Ni-NTA亲和层析纯化,梯度透析复性,SDS-PAGE分析纯度,Bradford法测定浓度;分别用5、10和50μg的NA加或不加MF59佐剂免疫BALB/c小鼠,检测其诱导产生NA特异性抗体滴度,评价免疫原性.结果 经一系列生物信息学软件分析得知,H5N1 NA为稳定的亲水性跨膜蛋白,无信号肽,二级结构以无规则卷曲和α螺旋为主,高级结构为四聚体结构,相对分子质量48 941.92,分子式C2148H3293N595O666S26,等电点6.13,不稳定系数31.84,总平均亲水性-0.268;经双酶切和测序验证,重组质粒pET-22b(+)-H5N1 NA构建成功,转化至E.coliBL21(DE3),经诱导表达、纯化、复性、浓缩获得重组NA纯度为94％,相对质量浓度为620.23 μg/mL,具有酶活性;使用该原核表达的NA免疫BALB/c小鼠,能产生结合H5N1疫苗原液中NA的特异性抗体,50 μg+MF59佐剂组平均效价达3 520.结论 原核表达的NA免疫小鼠诱导产生的抗体能够与甲型H5N1流感病毒天然的NA发生反应,为基于NA的流感重组亚单位疫苗研发提供了可行路径.

Objective:To express the head domain of influenza A virus hemagglutinin (HA) in a prokaryotic expression system and to evaluate its immunogenicity.Methods:The genes encoding the HA head domains of H1N1 and H3N2 influenza viruses were cloned into pET-22b(+ ) prokaryotic expression plasmid. After the induction with IPTG, the fusion proteins rH1N1-HA and rH3N2-HA containing HA head domain and His-tag were expressed and obtained from E. coli BL21. SDS-PAGE and Western blot was used to verify the expression of the recombinant proteins. Rabbits were immunized with multiple doses of the purified recombinant proteins to obtain polyclonal antibodies against the HA head domains of H1N1 and H3N2. The immunogenicity of the recombinant proteins was evaluated in BALB/c mice. Results:rH1N1-HA and rH3N2-HA induced protective antibodies (geometric mean titer ≥40) in mice and could be used as protective antigens. Polyclonal antibodies against rH1N1-HA and rH3N2-HA could be used as important materials for Western blot, ELISA and other immunological assays.Conclusions:The HA head domains prepared in this study could be used as protective antigens to induce protective antibodies in mice. Polyclonal antibodies against the HA head domains could be used for immunological and serological studies of influenza A viruses.

In influenza vaccine development, Madin-Darby canine kidney (MDCK) cells provide multiple advantages, including large-scale production and egg independence. Several cell-based influenza vaccines have been approved worldwide. We cultured H5N1 virus in a serum-free MDCK cell suspension. The harvested virus was manufactured into vaccines after inactivation and purification. The vaccine effectiveness was assessed in the Wuhan Institute of Biological Products BSL2 facility. The pre- and postvaccination mouse serum titers were determined using the microneutralization and hemagglutination inhibition tests. The immunological responses induced by vaccine were investigated using immunological cell classification, cytokine expression quantification, and immunoglobulin G (IgG) subtype classification. The protective effect of the vaccine in mice was evaluated using challenge test. Antibodies against H5N1 in rats lasted up to 8 months after the first dose. Compared with those of the placebo group, the serum titer of vaccinated mice increased significantly, Th1 and Th2 cells were activated, and CD8+ T cells were activated in two dose groups. Furthermore, the challenge test showed that vaccination reduced the clinical symptoms and virus titer in the lungs of mice after challenge, indicating a superior immunological response. Notably, early after vaccination, considerably increased interferon-inducible protein-10 (IP-10) levels were found, indicating improved vaccine-induced innate immunity. However, IP-10 is an adverse event marker, which is a cause for concern. Overall, in the case of an outbreak, the whole-virion H5N1 vaccine should provide protection.

目的 建立用于定量检测流感疫苗神经氨酸酶(neuraminidase,NA)含量的双抗体夹心ELISA方法,并进行验证.方法 采用多肽合成方式合成NA保守序列,经皮下多点免疫日本大耳白兔和豚鼠,共免疫5次,末次免疫后2周分别经颈动脉和心脏采血,分离血清,通过Protein G/A层析纯化,制备NA通用抗体.以鼠源通用抗体作为包被抗体,兔源通用抗体经HRP标记后作为酶标抗体,建立双抗体夹心ELISA法.确定包被抗体(32、16、8、4、2、1 μg/mL)的工作浓度及酶标抗体(1 ∶ 50～1 ∶ 6 400)的稀释度.验证方法的线性范围、准确度、重复性、中间精密度及耐用性.采用建立的方法测定流感裂解疫苗H1N1、H3N2、BV和BY型单价原液中的NA含量,并与荧光底物法进行比较.结果 兔源和鼠源通用抗体的效价分别为128 000和64 000,纯度分别为95％和96％,蛋白浓度分别为2 339和1 780μg/mL.建立双抗体夹心ELISA法的最佳包被抗体工作浓度为16μg/mL,最佳酶标抗体稀释度为1:400.NA(H3N2型)参考品在20～640 ng/mL浓度范围内与A450呈良好线性关系,R2均＞0.99;500、200、50 ng/mL的NA(H3N2型)参考品的平均样品回收率分别为102.15％、100.89％、100.70％,重复6次检测结果的CV均＜10％,2名实验员3次检测结果的CV均＜15％;不同抗原反应时间及酶标二抗孵育时间的样品回收率为85.41％～103.81％.建立的双抗体夹心ELISA法检测流感裂解疫苗H1N1、H3N2、BV、BY型单价原液NA含量分别为8.06、13.20、6.93、6.18 μg/mL,荧光底物法检测的NA活性分别为29 833、36 800、28 907、25 871 U/L,两者结果呈正相关(R2=0.979 2).结论 建立的NA含量双抗体夹心ELISA定量检测法具有良好的准确度、重复性、中间精密度和耐用性,可用于流感疫苗的检定及生产过程中对NA含量的质量控制.

目的 制备甲型H1N1流感病毒mRNA疫苗,并评价其免疫原性.方法 设计并构建分别含有甲型H1N1流感病毒血凝素(hemagglutinin,HA)和绿色荧光蛋白(enhanced green fluorescent protein,EGFP)基因的模板质粒,通过单酶切反应制备线性化转录模板,体外转录合成HA-mRNA和EGFP-mRNA.EGFP-mRNA体外转染HEK293T细胞,荧光显微镜下观察荧光表达情况.利用纳米药物制备系统制备HA-mRNA脂质体纳米颗粒(lipid nanoparticles,LNPs),即HA-mRNA-LNPs,测量粒径并计算包封率.用含2和10 μg HA-mRNA的HA-mRNA-LNPs经肌肉分别免疫BALB/c小鼠,同时设LNPs组(等体积未包裹mRNA的LNPs)和阴性对照组(等体积PBS),3周后进行加强免疫,免疫剂量和途径同上.加强免疫后3周,经小鼠眼眶后静脉丛采血,分离血清,采用血凝抑制(haemagglutination inhibition,HI)试验和病毒微量中和(microneutralization assay,MN)试验分别检测血凝抑制抗体滴度及中和抗体水平,并计算几何平均滴度(geometric average titer,GMT).结果 EGFP-mRNA转染HEK293T细胞后,镜下可见明显EGFP表达.HA-mRNA-LNPs的颗粒平均直径为70.2 nm,聚合物分散性指数为0.15,包封率约为80％.与阴性对照组比较,LNPs组小鼠血清的HI GMT和MN GMT差异均无统计学意义(P＞0.05),2和10 μg剂量组均显著升高(P均＜0.000 1);10 μg剂量组均显著高于2 μg剂量组(P均＜0.01).结论 制备的甲型H1N1流感病毒mRNA疫苗在BALB/c小鼠模型中具有较好的免疫原性,本研究为流感mRNA候选疫苗的研发提供了实验依据.

目的 探讨在小鼠模型中H5N1型流感病毒神经氨酸酶(neuraminidase,NA)的免疫原性,并进行攻毒试验评价其免疫保护作用.方法 制备H5N1型流感病毒裂解疫苗原液,磁珠法纯化获取NA蛋白.将BALB/c小鼠分为 2 μg NA、10 μg NA、50 μg NA、2 μg NA+MF59、10 μg NA+MF59、50 μg NA+MF59 和 MF59 组,共 7 组,每组10只,肌内注射免疫.初次免疫2周后,相同程序/剂量进行加强免疫,第4周采血分离血清,ELISA法测定效价;第5周进行攻毒试验,监测攻毒后2周内小鼠体重和体温变化,同时观察临床症状.结果 磁珠法纯化的H5N1 NA纯度达90％,浓度为201.23 μg/mL.2次免疫后,各剂量组血清效价呈剂量依赖性,加MF59佐剂组血清抗体效价均高于未加MF59佐剂组,其中50 μg NA+MF59组效价最高(平均效价为121 600).攻毒试验发现,50 μg NA+MF59能够保护100％(8只)的小鼠免受H5N1攻击,攻毒后50 μg NA+MF59组临床评分低于MF59组,但两组小鼠体温变化差异无统计学意义(P＞0.05).结论 免疫H5N1 NA能够诱导BLAB/c小鼠产生NA特异性抗体,使用MF59佐剂能够显著提高抗体效价,保护小鼠免受半数致死剂量的H5N1攻击,并能明显减轻临床症状.

Safe and effective vaccines against SARS-CoV-2 for children are urgently needed. Here we aimed to assess the safety and immunogenicity of an inactivated COVID-19 vaccine candidate, WIBP-CorV, in participants aged 3-17 years. A randomized, double-blind, placebo-controlled, phase 1/2 clinical trial was conducted in Henan Province, China, in healthy children aged 3-17 years. 240 participants in phase 1 trial and 576 participants in phase 2 trial were randomly assigned to vaccine or control with an age de-escalation in three cohorts (3-5, 6-12 and 13-17 years) and dose-escalation in three groups (2.5, 5.0 and 10.0μg/dose), and received 3 intramuscular injections at day 0, 28, and 56. WIBP-CorV showed a promising safety profile with approximately 17% adverse reactions within 30 days after injection and no grade 3 or worse adverse events. The most common adverse reaction was injection site pain, followed by fever, which were mild and self-limiting. The geometric mean titers of neutralizing antibody ranged from 102.2 to 1065.5 in vaccinated participants at 28 days after the third vaccination, and maintained at a range of 14.3 to 218.2 at day 180 after the third vaccination. WIBP-CorV elicited significantly higher titers of neutralizing antibody in the cohort aged 3-5 years than the other two cohorts. There were no detectable antibody responses in all alum-only groups. Taken together, our data demonstrate that WIBP-CorV is safe and well tolerated at all tested doses in participants aged 3-17 years, and elicited robust humoral responses against SARS-CoV-2 lasted for at least 6 months after the third vaccination. This study is ongoing and is registered with www.chictr.org.cn, ChiCTR2000031809.

Objective:To evaluate the immunogenicity of Madin-Darby canine kidney (MDCK) cell-based quadrivalent influenza split vaccine (MDCK-Va) combined with different adjuvants.Methods:Different doses of MDCK-Va and chicken embryo-based quadrivalent influenza split vaccine (egg-Va) were intramuscularly immunized BALB/c mice twice with an interval of three weeks. Serum samples were collected to detect antibody titers using hemagglutination inhibition (HI) assay. BALB/c mice were immunized with different doses of MDCK-Va combined with QS21, AddVax, PolyI∶C, CpG ODN 1826 and AddVax/PolyI∶C (Add/Poly), respectively. HI and microneutralization assays were used to detect antibody titers 21 d after the first and booster immunization. Spleen tissues were collected from the mice immunized with 10 μg MDCK-Va combined with the above adjuvants 5 d after the booster immunization to analyze spleen index and the types of spleen cells.Results:The immunoprotective effect of MDCK-Va was not inferior to that of egg-Va. MDCK-Va combined with each of the above adjuvants could induce higher HI antibody titer than MDCK-Va alone, especially the QS21/Va and Add/Poly/Va groups, and the differences were statistically significant. For H1N1 vaccine, the Pearson′s correlation coefficient ( r) between HI antibody and neutralizing antibody was 0.737-0.910, and for H3N2 subtype vaccine, the value of r was 0.839-0.947. Compared with the MDCK-Va group, the QS21/Va group showed significantly increased spleen index and decreased proportion of single lymphocytes. QS21 and Add/Poly were much better than other adjuvants in stimulating mouse splenic neutrophils and CD4/CD8 cells. Conclusions:Add/Poly had a stronger immune enhancement effect on MDCK-Va, suggesting that it was a potential adjuvant for MDCK-Va. The antibody titer detected by HI and MN assays had a strong positive correlation.

目的 真核表达H1N1型流感病毒血凝素(hemagglutinin,HA)蛋白胞外段,并分析其免疫原性.方法 以H1N1型流感毒株基因组为参考序列,将H1N1 HA的胞外段氨基酸序列进行密码子优化后基因合成,将合成的目的 基因片段与KS001载体连接,构建重组质粒KS001/HA,转染至Expi293F真核细胞,收集表达产物,进行12％ SDS-PAGE、Western blot及N-糖基化鉴定;将表达产物经Capto Q离子交换层析柱纯化,收集纯化产物进行12％ SDS-PAGE鉴定,BCA法测定蛋白浓度;用不同浓度的HA胞外段蛋白辅以佐剂免疫小鼠,通过病毒微量中和试验(mi-croneutralization test,MNT)检测小鼠血清抗体效价.结果 经菌液PCR、双酶切及测序鉴定证明质粒构建正确;表达的重组蛋白以单体形式存在,相对分子质量约70000,N-糖基化丰富;重组蛋白与佐剂配伍后的各剂量组均产生针对H1N1型病毒特异性抗体,且阳转率≥90％.结论 成功构建了重组质粒KS001/HA,并于真核细胞中表达,表达产物联合佐剂免疫小鼠后具有较好的免疫原性.

流行性感冒(简称流感)是一种由流感病毒引起的急性呼吸道传染病,据WHO统计,每年季节性流感可造成约65万人死亡,接种疫苗是预防流感最有效的方法.神经氨酸酶(neuraminidase,NA)是流感裂解疫苗的主要抗原之一,可够诱导机体产生NA特异性抗体,该抗体在预防流感病毒感染、抑制病毒扩散、减轻临床症状及增强疫苗效力方面发挥重要作用.NA的基因相对保守,且NA特异性抗体具有广泛交叉反应,是通用流感疫苗的理想抗原.基于NA流感疫苗广阔的应用前景,本文就NA的结构及功能、NA基因的相对保守性、NA的免疫原性及免疫机制、基于NA的流感疫苗、NA流感疫苗面临的挑战等作一综述.

Objective:To establish a method for isolation and purification of neuraminidase from influenza vaccine and to prepare reference substance for quantitative detection of neuraminidase.Methods:Functional magnetic particles with specific affinity for neuraminidase were prepared. The method for separation and purification of neuraminidase was established based on the magnetic particles. The separation and purification conditions were optimized. The purity of neuraminidase was analyzed and the specificity was verified. The enzyme activity was determined and the protein was quantified.Results:The functional magnetic particles modified with 4-aminophenanthroline were successfully prepared and the method for isolation and purification of neuraminidase based on the magnetic particles was established. The purity of neuraminidase was 98.7%. The concentrations of neuraminidase isolated and purified from the monovalent stock solution of H1N1, H3N2, B/Victoria and B/Yamagate vaccines were 71.50, 100.58, 64.11 and 37.68 μg/ml, respectively, and the enzyme activity remained.Conclusions:The method for isolation and purification of influenza virus neuraminidase was established and the corresponding reference substance was prepared.

新型冠状病毒肺炎(Coronavirus Disease 2019,COVID-19)是由重症急性呼吸综合征冠状病毒2(severe acute respiratory syndrome coronavirus 2,SARS-CoV-2)感染引起的呼吸道传染病.COVID-19在全球范围的大流行造成了严重的公共卫生危机,已成为导致死亡的主要原因之一.为减轻COVID-19对公共健康和社会经济的影响,全球疫苗研发机构均在积极开发疫苗,目前上市的COVID-19疫苗包括灭活疫苗、病毒载体疫苗、mRNA疫苗和重组蛋白疫苗等多种类型.传统经典的灭活疫苗由于其生产工艺成熟、安全性良好、免疫原性稳定、可及性强,受到众多疫苗企业的青睐.目前已有多款COVID-19灭活疫苗获得紧急使用或批准附条件上市.本文就COVID-19灭活疫苗研发的毒种与细胞基质选择、生产工艺、临床前研究、对突变株的保护作用作一综述,并着重探讨COVID-19灭活疫苗的临床研究进展及其面临的挑战.