ABSTRACT Seasonal influenza, causes hundreds of thousands of deaths annually, posing a severe threat to human health. Currently available influenza vaccines are targeted only at specific strains or conserved epitopes; however, these vaccines are not completely efficacious because influenza viruses can undergo mutation during circulation, leading to antigenic mismatch between recommended strains and circulating strains and elusion from the immune system. Therefore, developing an influenza vaccine that is quick, effective, and broadly protective has become crucial, and the integral part of hemagglutinin (HA) remains an ideal target for vaccine development. This study developed a lipid nanoparticle-encapsulated nucleoside-modified mRNA vaccine (mRNA-LNPs) encoding a consensus full-length HA sequence (H1c) and evaluated its protective efficacy and immunogenicity through in vitro and in vivo assays. Following two intramuscular immunizations (2, 10 µg, or 20 µg) at a 3-week interval in BALB/c mice, H1c-mRNA-LNP vaccine induced strong antibodies as shown in the hemagglutination-inhibition test and protective neutralizing antibodies against numerous heterologous H1N1 influenza viruses as shown in the microneutralization assay. Additionally, both Th1- and Th2-biased cellular immune responses were elicited, with the Th1-biased response being stronger. Two doses of the H1c-mRNA-LNP vaccine could neutralize a panel of heterologous H1N1 influenza viruses and could confer protection in mice. Taken together, these findings suggest that the H1c-mRNA-LNP vaccine encoding a consensus full-length HA is a feasible strategy for developing a cross-protective vaccine against a panel of heterologous H1N1 influenza viruses.

Objective:To prepare a recombinant hemagglutinin trimer (HA-Tri) vaccine against influenza viruses and to study its immunogenicity in a mouse model.Methods:A stable CHO cell line that could express HA-Tri was constructed. Western blot, single radial immunodiffusion, protein particle size detection and N-glycosylation site analysis were performed for qualitative and quantitative analysis of the recombinant protein. According to the different treatment conditions such as dosage and adjuvant, BALB/c mice were divided into 11 groups and subjected to consistent immunization procedures. Serum neutralizing antibody titers were measured on 56 d after the first immunization to evaluate the immunogenicity of HA-Tri.Results:The constructed CHO cells could secret and express HA-Tri proteins. The HA-Tri proteins were biologically active and capable of forming precipitation rings in the single radial immunodiffusion. The particle size of HA-Tri was approximately 18.79 nm and 10 N-glycosylation sites were detected, including high mannose, complex glycoforms and heterozygous glycoforms. After prime-boost immunization, there was no statistically significant difference in the titers of neutralizing antibodies induced in mice by 3.75 μg of HA-Tri in combination with RFH01 adjuvant and 15 μg of monovalent vaccine stock solution ( P=0.431 2, U=36). Serum antibody titers in the HA-Tri+ RFH01 groups were higher than those in the corresponding HA-Tri groups without RFH01 adjuvant, and the highest titer was induced in the 15 μg HA-Tri+ RFH01 group, which was 1 280. Conclusions:The recombinant HA-Tri protein was successfully prepared. HA-Tri in combination with RFH01 adjuvant could induce humoral immune responses against influenza viruses in BALB/c mice, which would provide reference for the development of influenza virus recombinant subunit vaccines.

Tumor necrosis factor receptor-associated factor 3 (TRAF3), an adaptor protein, has significant and varying effects on immunity depending on cell types. The role of TRAF3 in Madin-Darby Canine Kidney Epithelial (MDCK) cell resistance to influenza A virus (IVA) remains elusive. In the present study, CRISPR-Cas9 gene editing technology was used to construct the TRAF3 knockout MDCK cells (MDCK-TRAF3- /- ). Hemagglutination assay, plaque assay, transcriptome, and quantitative real-time PCR were performed after IVA infection. The results showed that after IVA infection, HA titers and virus titers were promoted, interferon I-related pathways were significantly blocked, and transcription of several antiviral-related genes was significantly decreased in MDCK-TRAF3- /- cells. Thus, our study suggests that TRAF3 gene knockout reduced MDCK cell's resistance to IVA, thereby resulting in a promising way for IVA isolation and vaccine manufacturing.

Objective:To evaluate the immunogenicity of a quadrivalent subunit vaccine combined with RFH01 adjuvant in a mouse model.Methods:Identification tests were performed on four monovalent influenza virus subunit vaccine stock solutions according to the methods described in Part 3 of the Chinese Pharmacopoeia 2020 Edition. In the study of the quadrivalent subunit vaccine combined with RFH01 adjuvant, 460 female BALB/c mice (6-8 weeks old) were randomly divided into 46 groups including experimental groups, vaccine control group, negative control group and blank group with 10 mice in each group. In the study of the quadrivalent subunit vaccine in old and young mice, 80 female 10-month-old and 80 female 10-week-old BALB/c mice were randomly divided into 16 groups ( n=10) including monovalent influenza virus vaccine group, quadrivalent subunit vaccine group, quadrivalent subunit vaccine+ RFH01 adjuvant group, chicken embryo quadrivalent split vaccine control group and PBS group. All mice were immunized by intramuscular injection. At 21 d after the primary immunization, a booster immunization was conducted using the same strategy. Blood samples were collected at 21 d and 42 d after the primary immunization for serum separation. Haemagglutination inhibition (HI) test was performed to detect the antibody levels in mouse serum samples. Results:After the booster immunization, the positive conversion rates in all vaccine+ RFH01 adjuvant groups reached 100%, and the geometric mean titers (GMTs) of serum antibodies were significantly higher than those of the vaccine groups without RFH01 adjuvant. There were significant differences in serum antibody titers between the monovalent/quadrivalent subunit vaccine groups with and without RFH01 adjuvant. After the booster immunization, the titers of serum antibodies against H1N1, H3N2, B/Victoria and B/Yamagata in the 10-week-old mice were significantly higher than those in the 10-month-old mice.Conclusions:The monovalent and quadrivalent influenza virus vaccines in combination with RFH01 adjuvant could elicit higher antibody titers in young (6-10 weeks old) and old (10 months old) mice, showing good immunogenicity.

Gene therapy is a technique involving the modification of an individual's genes for treating a particular disease. The key to effective gene therapy is an efficient carrier delivery system. Viral vectors that have been artificially modified to lose their pathogenicity are used widely as a delivery system, with the key advantages of their natural high transduction efficiency and stable expression. With decades of development, viral vector-based gene therapies have achieved promising clinical outcomes. Currently, the three key vector strategies are based on adeno-associated viruses, adenoviruses, and lentiviruses. However, certain challenges, such as immunotoxicity and "off-target", continue to exist. In the present review, the above three viral vectors are discussed along with their respective therapeutic applications. In addition, the major translational challenges encountered in viral vector-based gene therapies are summarized, and the possible strategies to address these challenges are also discussed.

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.

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.

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.