Human immune responses elicited by an intranasal inactivated H5 influenza vaccine

Intranasally administered influenza vaccines could be more effective than injected vaccines, because intranasal vaccination can induce virus-specific immunoglobulin A (IgA) antibodies in the upper respiratory tract, which is the initial site of infection. In this study, immune responses elicited by an intranasal inactivated vaccine of influenza A(H5N1) virus were evaluated in healthy individuals naive for influenza A(H5N1) virus. Three doses of intranasal inactivated whole-virion H5 influenza vaccine induced strong neutralizing nasal IgA and serum IgG antibodies. In addition, a mucoadhesive excipient, carboxy vinyl polymer, had a notable impact on the induction of nasal IgA antibody responses but not on serum IgG antibody responses. The nasal hemagglutinin (HA)-specific IgA antibody responses clearly correlated with mucosal neutralizing antibody responses, indicating that measurement of nasal HA-specific IgA titers could be used as a surrogate for the mucosal antibody response. Furthermore, increased numbers of plasma cells and vaccine antigen-specific Th cells in the peripheral blood were observed after vaccination, suggesting that peripheral blood biomarkers may also be used to evaluate the intranasal vaccine-induced immune response. However, peripheral blood immune cell responses correlated with neutralizing antibody titers in serum samples but not in nasal wash samples. Thus, analysis of the peripheral blood immune response could be a surrogate for the systemic immune response to intranasal vaccination but not for the mucosal immune response. The current study suggests the clinical potential of intranasal inactivated vaccines against influenza A(H5N1) viruses and highlights the need to develop novel means to evaluate intranasal vaccine-induced mucosal immune responses.     

Efficient disulfide bond formation in virus-like particles

Virus-like particles (VLPs) consist of a virus's outer shell but without the genome. Similar to the virus, VLPs are monodisperse nano-capsules which have a known morphology, maintain a high degree of symmetry, and can be engineered to encapsidate the desired cargo. VLPs are of great interest for vaccination, drug/gene delivery, imaging, sensing, and material science applications. Here we demonstrate the ability to control the disulfide bond formation in VLPs by directly controlling the redox potential during or after production and assembly of VLPs. The open cell-free protein synthesis environment, which has been reported to produce VLPs at yields comparable or greater than traditional in vivo technologies, was employed. Optimal conditions for disulfide bond formation were found to be VLP dependent, and a cooperative effect in the formation of such bonds was observed.     

流感疫苗研制的新策略

流感病毒感染可引起急性呼吸道传染病,严重危害人类的健康与生命。疫苗免疫是防控流感的重要手段。目前广泛应用的传统灭活疫苗和减毒活疫苗,在预防流感中发挥了重要作用,但存在通用性差和免疫效率低等不足。研制更为安全高效特别是能针对多种流感亚型的新型广谱疫苗成为当前流感疫苗研究的热点。随着结构生物学和反向遗传生物学等新技术的迅速发展,一些新策略不断应用于新型流感疫苗的研究,显示出良好的应用前景。     

广州地区GⅡ.17型诺如病毒GZ-L343毒株病毒样颗粒的构建与免疫特征的鉴定

【背景】人源诺如病毒是急性胃肠炎暴发的主要原因,GII.4是过去几十年的主要流行基因型。2014/2015年出现的GII.17型变异株是中国首例导致大规模暴发的非GII.4流行株。通过对来自华南地区的诺如病毒GII.17型毒株的完整基因组序列进行分析,证实了该GII.17型突变株与先前确定的GII型变异株不同。【目的】制备广州地区GII.17型诺如病毒GZ-L343的病毒样颗粒,并系统表征其免疫原性及功能特性。【方法】借助杆状病毒表达系统制备GII.17-GZ-L343的病毒样颗粒,并通过氯化铯梯度超速离心对其进行纯化,制备抗血清并对其免疫功能进行评价。【结果】聚丙烯酰胺凝胶电泳和蛋白质免疫印迹结果表明所得蛋白分子量大小约为58kDa;透射电镜结果表明病毒样颗粒直径约为30nm;酶联免疫吸附测定结果显示该病毒样颗粒具有较好的免疫原性;唾液组织血型抗原的体外受体结合测定表明,该病毒样颗粒与部分A型、B型、O型及AB型分泌及非分泌血型样本存在阳性结合;效价测定结果表明免疫所得血清效价在104以上;交叉反应结果表明该抗血清与异型病毒样颗粒不存在交叉反应。此外,体外阻断结果表明,该抗血清仅能阻...     

Self-assembly of sapovirus recombinant virus-like particles from polyprotein in mammalian cells

The SaV genome is a positive-sense, non-segmented single-strand RNA molecule of approximately 7.5 kb that is polyadenylated at its 3' terminus. The major capsid (VP1) of SaV is thought to be produced as the ORF1 polyprotein followed by cleavage, or translation from subgenomic RNA (3'-coterminal with the virus genome), or both. We have recently reported the formation of SaV VLP from subgenomic-like RNA in mammalian cells. In the present study, we demonstrated that the VP1 cleaved from a part of ORF1 polyprotein self-assembled into VLP in mammalian cells when a transient expression system using a recombinant vaccinia virus encoding T7 RNA polymerase was used.     

Incorporation of dengue virus replicon into virus-like particles by a cell line stably expressing precursor membrane and envelope proteins of dengue virus type 2

While virus-like particles (VLPs) containing subgenomic replicons, which can transduce replicons into target cells efficiently for studying viral replication and vectors of gene therapy and vaccine, have been established for several flaviviruses, none has been reported for the four serotypes of dengue virus, the causal agent of the most important arboviral diseases in this century. In this study, we successfully established a cell line stably expressing the precursor membrane/envelope (PrM/E) proteins of dengue virus type 2 (DENV2), which can package a DENV2 replicon with deletion of PrM/E genes and produce single-round infectious VLPs. Moreover, it can package a similar replicon of different serotype, dengue virus type 4, and produce infectious chimeric VLPs. To our knowledge, this study reports for the first time replicon-containing VLPs of dengue virus. Moreover, this convenient system has potential as a valuable tool to study encapsidation of dengue virus and to develop novel chimeric VLPs containing dengue virus replicon as vaccine in the future.     

H5N1重组禽流感病毒样颗粒免疫原性研究

目的对构建的H5N1重组禽流感病毒样颗粒(VLPs)进行初步免疫原性探讨,并与H5N1全病毒灭活疫苗(WIV)进行体液免疫和细胞免疫的比较。方法在0周和3周分别以纯化H5N1重组禽流感病毒样颗粒、H5N1全病毒灭活疫苗及pH7.2 PBS腿部肌肉注射BALB/c小鼠,于不同时间收集血清,以血凝抑制试验(HI)和血清IgG抗体酶联免疫吸附试验(ELISA)评估体液免疫,CD4+、CD8+T细胞亚群及酶联免疫斑点试验(ELISPOT)评估细胞免疫,并以同型毒株滴鼻攻击,观察小鼠存活率。结果病毒样颗粒各组和全病毒灭活疫苗免疫后小鼠血清ELISA IgG效价均有升高;中和抗体效价除病毒样颗粒120 ng/只免疫剂量外其他免疫小鼠HI效价均达1︰40;小鼠脾CD4+T淋巴细胞亚群分类:全病毒灭活疫苗组(600μg/只)为36.56%;病毒样颗粒组(120 ng/只,600 ng/只,2 500 ng/只)分别为26.58%,32.20%,29.25%;PBS组为26.65%;CD8+T淋巴细胞亚群分类:全病毒灭活疫苗组(600 ng/只)为10.78%;病毒样颗粒组(120 ng/只,600 ng/只,2 500 ng/只)分别为1 3.53%,14.24%,1 3.35%;PBS组为10.69%。ELISPOT试验统计学数据显示,病毒样颗粒和全病毒灭活疫苗的小鼠脾单个核细胞分泌IFN-γ细胞与PBS组有显著性差异;小鼠保护性试验结果显示,除病毒样颗粒120 ng/只免疫剂量小鼠的存活率为87.5%外,其他病毒样颗粒实验组小鼠均为100%,PBS对照组为12.5%。结论 H5N1重组禽流感病毒样颗粒能诱导体液免疫和细胞免疫,并能抵御同型病毒株的攻击,可作为H5N1人用禽流感的候选疫苗。     

Influenza virus‐like particles produced by transient expression in Nicotiana benthamiana induce a protective immune response against a lethal viral challenge in mice

A strain‐specific vaccine represents the best possible response to the threat of an influenza pandemic. Rapid delivery of such a vaccine to the world's population before the peak of the first infection wave seems to be an unattainable goal with the current influenza vaccine manufacturing capacity. Plant‐based transient expression is one of the few production systems that can meet the anticipated surge requirement. To assess the capability of plant agroinfiltration to produce an influenza vaccine, we expressed haemagglutinin (HA) from strains A/Indonesia/5/05 (H5N1) and A/New Caledonia/20/99 (H1N1) by agroinfiltration of Nicotiana benthamiana plants. Size distribution analysis of protein content in infiltrated leaves revealed that HA was predominantly assembled into high‐molecular‐weight structures. H5‐containing structures were purified and examination by transmission electron microscopy confirmed virus‐like particle (VLP) assembly. High‐performance thin layer chromatography analysis of VLP lipid composition highlighted polar and neutral lipid contents comparable with those of purified plasma membranes from tobacco plants. Electron microscopy of VLP‐producing cells in N. benthamiana leaves confirmed that VLPs accumulated in apoplastic indentations of the plasma membrane. Finally, immunization of mice with two doses of as little as 0.1 µg of purified influenza H5‐VLPs triggered a strong immune response against the homologous virus, whereas two doses of 0.5 µg of H5‐VLPs conferred complete protection against a lethal challenge with the heterologous A/Vietnam/1194/04 (H5N1) strain. These results show, for the first time, that plants are capable of producing enveloped influenza VLPs budding from the plasma membrane; such VLPs represent very promising candidates for vaccination against influenza pandemic strains.     

Screen of multifunctional monoclonal antibodies against hepatitis B core virus-like particles

HBc-VLP can be used in an epitope presentation system to carry foreign epitopes and mimic live virus in order to study viral particle uptake, virion-mediated activation and antigen presentation by dendritic cells. In this study, a multifunctional mAb was produced using a novel research strategy. A truncated HBc-VLP bone vector with a special conformation was used as an immunogen and the target hybridoma cell lines were screened by a series of tests; including ELISA, Western blot, and cellular immunofluorescence based on the epitope presentation system. The screened monoclonal antibody was used to identify the HBc-VLP vector, a fusion HBc-VLP vaccine, and intracellular HBV capsids. The new strategy facilitated acquisition of the desired mAbs and will serve as a reference for other VLP-related research.     

NADC30-like猪繁殖与呼吸综合征病毒样颗粒的制备与鉴定

【背景】猪繁殖和呼吸综合征病毒(porcine reproductive and respiratory syndrome virus,PRRSV)可以造成怀孕母猪的繁殖障碍及仔猪的呼吸系统疾病,近年来,NADC30-like谱系PRRSV已成为国内的优势流行毒株。【目的】研制针对NADC30-like谱系PRRSV的病毒样颗粒(virus-like particle,VLP)疫苗。【方法】将PRRSV NADC30-like毒株编码GP5蛋白开放阅读框5(open reading frame 5,ORF5)、ORF6(编码M蛋白)分别连接至pFastBac^TMDual载体P₁₀和P_H启动子下游多克隆位点,获得穿梭质粒pFB-30-ORF5及pFB-30-ORF6,酶切鉴定后,将ORF6基因插入到穿梭质粒pFB-30-ORF5 P_H启动子下游,构建穿梭质粒pFB-30-ORF5-OPF6。将上述3种穿梭质粒分别转化大肠杆菌DH10Bac感受态细胞,通过蓝白斑筛选及PCR鉴定重组杆粒。再将获得的重组杆粒转染至SF9昆虫细胞,发现细胞病变后收获病毒液,继续盲传3代,在透射电镜下观察是否有病毒样颗粒。用第3代病毒液感染SF9细胞后,分别用GP5蛋白、His-tag、Flag-tag单克隆抗体作为一抗,通过免疫电镜、间接免疫荧光(indirect immunofluorescence assay,IFA)、Western blotting鉴定重组蛋白。【结果】成功构建了3种穿梭质粒pFB-30-ORF5、pFB-30-ORF6和pFB-30-ORF5-OPF6,酶切鉴定正确。通过蓝白斑筛选及PCR验证后获得重组杆粒,分别命名为Bacmind-30-ORF5、Bacmind-30-ORF6和Bacmind-30-ORF5-ORF6。重组杆粒感染SF9细胞120h时出现明显的细胞病变,收获病毒液后,在透射电子显微镜可观察到大小为50nm左右呈现球形结构的VLPs。免疫电镜可以观察到胶体金颗粒结合在VLPs周围;IFA结果显示实验组均出现了明显绿色的特异性荧光灶;Western blotting结果表明,3种VLPs均出现特异性条带,并与预期大小一致。【结论】制备了3种NADC30-like谱系PRRSV的病毒样颗粒,为针对PRRSV新谱系流行株疫苗的研发奠定了基础。     

A quantitative description of in vitro assembly of human papillomavirus 16 virus-like particles

The full-length human papillomavirus 16 major capsid protein L1 is expressed in Saccharomyces cerevisiae as virus-like particles (VLPs). However, yeast-expressed human papillomavirus 16 particles are irregular in shape and are prone to aggregate. When disassembled and reassembled, the resulting particles have improved stability and solubility. We have examined VLP dissociation and reassembly to define the important features of the assembly mechanism. We found that the VLPs rapidly disassemble at pH 8.2 and low ionic strength in the presence of low concentrations of reducing agents. The pH dependence of assembly kinetics and extent of assembly under reducing conditions were differentially sensitive to ionic strength. Assembly at pH 5.2 was very fast and led to heavily aggregated particles. This sort of kinetic trap is expected for overinitiated assembly. We observed that reassembly at pH 6.2, 7.2, and 8.2 yielded regular particles over a broad range of ionic strength. At these three pH values, assembly was quantitative at 1 M NaCl. At pH 7.2, much more than at pH 6.2 or pH 8.2, assembly decreased monotonically with ionic strength. The free energy of association ranged from − 8 to − 10 kcal/mol per pentamer. The effect of pH on assembly was further investigated by examining dissociation of reassembled particles. Though indistinguishable by negative stain electron microscopy, particles assembled at pH 7.2 disassembled slower than pH 5.2, 6.2, or 8.2 VLPs. We hypothesize that pH 7.2 assembly reactions lead to formation of particles with conformationally different interactions.     

牛病毒性腹泻病毒1型病毒样颗粒的制备及其对豚鼠的免疫原性分析

为了获得预防牛病毒性腹泻病毒1型(bovine viral diarrhea virus 1,BVDV-1)感染的病毒样颗粒,扩增C-Ems-E1-E2编码区段并克隆至pFastBacDaul载体,转化大肠杆菌DH10Bac感受态细胞与Bacmid重组获得Bacmid-BVDV-1,转染至Sf9细胞,获得重组杆状病毒B...     

In vitro self-assembled HCV core virus-like particles induce a strong antibody immune response in sheep.

The in vitro self-assembly properties of the entire hepatitis C virus core protein (HCcAg) obtained from Pichia pastoris cells and the induction of specific antibody immune response were studied. HCcAg was purified as a low-molecular-weight species by electroelution under denaturing conditions for confirmation of its self-assembly properties. After renaturalization, electron microscopy showed that HCcAg assembled into spherical particles of 30 nm. HCcAg also showed homogeneity and was specifically recognized by serum from a chronic HCV carrier patient. The data indicated that in vitro assembly of HCcAg, into virus-like particles resembling HCV nucleocapsid particles at a mature stage, is an intrinsic quality of this protein. Finally, HCcAg generated a strong antibody immune response in sheep, suggesting its usefulness for stimulating the host immune response against HCV.     

Generation of Hepatitis B virus PreS2-S antigen in Hansenula polymorpha

Despite the long availability of a traditional prophylactic vaccine containing the HBV surface antigen(HBsA g) and aluminum adjuvant, nearly 10% of the population remains unable to generate an effective immune response. Previous studies have indicated that hepatitis B virus(HBV) PreS 2-S is abundant in T/B cell epitopes, which induces a stronger immune response than HBsA g, particularly in terms of cytotoxic T lymphocyte(CTL) reaction. In the current study, the HBV PreS 2-S gene encoding an extra26 amino acids(PreS 2 C-terminus) located at the N-terminus of HBsA g was cloned into the pV CH1300 expression vector. Pre S2-S expressed in the methylotrophic yeast, Hansenula polymorpha, was produced at a yield of up to 250 mg/L. Subsequent purification steps involved hydrophobic adsorption to colloidal silica, ion-exchange chromatography and density ultracentrifugation. The final product was obtained with a total yield of ~15% and purity of ~99%. In keeping with previous studies, ~22 nm viruslike particles were detected using electron microscopy. The generated PreS 2-S antigen will be further studied for efficacy and safty in animals.     

Peptide epitope identification by affinity selection on bacteriophage MS2 virus-like particles

Filamentous phages are now the most widely used vehicles for phage display and provide efficient means for epitope identification. However, the peptides they display are not very immunogenic because they normally fail to present foreign epitopes at the very high densities required for efficient B-cell activation. Meanwhile, systems based on virus-like particles (VLPs) permit the engineered high-density display of specific epitopes but are incapable of peptide library display and affinity selection. We developed a new peptide display platform based on VLPs of the RNA bacteriophage MS2. It combines the high immunogenicity of MS2 VLPs with the affinity selection capabilities of other phage display systems. Here, we describe plasmid vectors that facilitate the construction of high-complexity random sequence peptide libraries on MS2 VLPs and that allow control of the stringency of affinity selection through the manipulation of display valency. We used the system to identify epitopes for several previously characterized monoclonal antibody targets and showed that the VLPs thus obtained elicit antibodies in mice whose activities mimic those of the selecting antibodies.     

乙肝病毒核心蛋白病毒样颗粒表面抗原密度对抗体应答水平的影响

【目的】为了探究乙肝病毒核心蛋白(HepatitisBviruscoreprotein,HBc)病毒样颗粒(Virus-like particles,VLPs)表面抗原密度对免疫后抗体应答水平的影响,制备了不同抗原密度的HBc VLPs疫苗,并检测了其在小鼠体内的抗体应答水平。【方法】首先制备了N端带有3个甘氨酸的人巨细胞病毒重组抗原域AD-4作为模式抗原,接着通过Sortase A的介导将AD-4连接到HBc VLPs表面上。将系列浓度梯度AD-4抗原在SortaseA介导下分别与相同浓度的HBcVLPs发生反应,制备不同抗原密度的HBc-AD-4 VLPs。将其分别免疫6–8周龄BALB/c小鼠3次,每次免疫间隔2 w,间接ELISA法检测被免疫小鼠血清的抗体应答水平。【结果】结果表明,当HBc VLPs表面抗原密度为44.4%时,即HBc反应浓度∶AD-4反应浓度为1:0.5时,不足以引起高滴度的抗体产生;当HBc VLPs表面抗原密度为64.2%时,即HBc反应浓度∶AD-4反应浓度为1:1时,HBc-AD-4 VLPs诱导的AD-4特异性抗体滴度与100%抗原密度的HBc-AD-4VLPs所引起的抗体滴度相当;当HBcVLPs表面抗原密度大于64.2%时,引起的抗体应答水平不因抗原密度增加而进一步增强。【结论】发现了HBcVLPs表面抗原密度与免疫后抗体滴度呈正相关,然而免疫64.2%抗原密度的HBc VLPs所产生的抗体滴度可达峰值,抗原密度进一步增加,抗体应答水平不会进一步加强。     

Production of CCHF virus-like particle by a baculovirus-insect cell expression system

Crimean-Congo Haemorrhagic Fever Virus (CCHFV) is a tick-born virus of the Nairovirus genus within the Bunyaviridae family, which is widespread and causes high fatality. The nucleocapsid of CCHFV is comprised of N proteins that are encoded by the S segment. In this research, the N protein of CCHFV was expressed in insect cells using a recombinant baculovirus. Under an electron microscope, Virus-Like Particles (VLPs) with various size and morphology were observed in cytoplasmic vesicles in the infected cells. Sucrose-gradient purification of the cell lysate indicated that the VLPs were mainly located in the upper fraction after ultracentrifugation, which was confirmed by Western blot analysis and immuno-electron microscopy (IEM).     

Effect of MOI ratio on the composition and yield of chimeric infectious bursal disease virus-like particles by baculovirus co-infection: deterministic predictions and experimental results

Virus-like particles (VLPs) are empty particles consisting of virus capsid proteins that closely resemble native virus but are devoid of the native viral nucleic acids and therefore have attracted significant attention as noninfectious vaccines. A recombinant baculovirus, vIBD-7, which encodes the structural proteins (VP2, VP3, and VP4) of infectious bursal disease virus (IBDV), produces native IBD VLPs in infected Spodoptera frugiperda insect cells. Another baculovirus, vEDLH-22, encodes VP2 that is fused with a histidine affinity-tag (VP2H) at the C-terminus. By co-infection with these two baculoviruses, hybrid VLPs with histidine tags were formed and purified by immobilized metal affinity chromatography (Hu et al., 1999). Also, we demonstrated that varying the MOI ratio of these infecting viruses altered the extent of VP2H incorporated into the particles. A dynamic mathematical model that described baculovirus infection and VLP synthesis (Hu and Bentley, 2000) was adapted here for co-infection and validated by immunofluorescence labeling. It was shown to predict the VLP composition as a dynamic function of MOI. A constraint in the VP2H content incorporated into the particles was predicted and shown by experiments. Also, the MOI ratio of both infecting viruses was shown to be the major factor influencing the composition of the hybrid particles and an important factor in determining the overall yield. ELISA results confirmed that VP2H was exhibited to a varied extent on the outer surface of the particles. This model provides insight on the use of virus co-infection in virus-mediated recombinant protein expression systems and aids in the optimization of chimeric VLP synthesis.     

共表达H5N1流感病毒M1和HA基因的DNA疫苗与腺病毒载体疫苗的小鼠免疫评价

为评价在小鼠体内表达流感病毒M1和HA基因诱导的免疫反应,制备共表达H5N1亚型禽流感病毒 (A/Anhui/1/2005) 全长基质蛋白1 (M1) 基因和血凝素 (HA) 基因的重组DNA疫苗pStar-M1/HA和重组腺病毒载体疫苗Ad-M1/HA,将其按初免-加强程序免疫BALB/c小鼠,共免疫4次,每次间隔14 d。第1、3次用DNA疫苗,第2、4次用重组腺病毒载体疫苗,每次免疫前及末次免疫后14 d采集小鼠血清用于检测体液免疫应答,末次免疫后14 d采集小鼠脾淋巴细胞用于检测细胞免疫应答。血凝     

Genetic characterization of swine influenza viruses isolated in Japan between 2009 and 2012

Eleven swine influenza viruses (SIVs) isolated from pigs in Japanese institutions between 2009 and 2012 were genetically characterized. Seven H1N1 were shown to have originated from A(H1N1)pdm09 viruses. Two H1N2 viruses contained H1 and N2 genes of Japanese H1N2 SIV origin together with internal genes of A(H1N1)pdm09 viruses. Two H3N2 viruses isolated during animal quarantine were identified as triple reassortant H3N2 viruses maintained among pigs in North America. This study shows that A(H1N1)pdm09 viruses and their reassortant strains are already present in domestic pigs in Japan and that novel SIVs are possibly being imported from abroad.