四价流感病毒裂解疫苗B型血凝素含量的检测方法

目的建立能准确测定四价流感病毒裂解疫苗中B型血凝素含量(单向免疫扩散法,SRD)的检测方法。方法采用双价抗原参考品SRD法对四价流感病毒裂解疫苗中两种B型血凝素含量进行测定。将B1与B2抗原参考品按质量浓度1∶1混合制备为双价抗原参考品,双价抗原和待检样品与10%裂解剂按9∶1比例裂解30 min,分别加入到含有抗血清参考品的1.5%琼脂糖凝胶板上,每孔10μL,置20~25℃放置18~24 h,经干燥、染色、脱色,测量结果。验证双价抗原SRD法的重复性和准确性。结果双价抗原SRD法测定的血凝素含量平均值比单价抗原SRD法更接近理论配制值,故双价抗原SRD法比单价抗原SRD法能更能准确检测QIV中两种B型血凝素含量,经验证双价抗原SRD法的重复性及准确性良好。结论双价抗原SRD法提高了四价流感病毒裂解疫苗B型血凝素含量检测的准确性,为精确测定四价流感病毒裂解疫苗中B型血凝素含量提供了有效的方法和数据支持。     

流感病毒DNA疫苗的研究进展

流感病毒是一种危害极大的病原体。常见的流感疫苗有灭活疫苗、减毒活疫苗。随着分子生物学技术的发展,流感DNA疫苗成为流感疫苗的一个重要发展方向。常见的流感DNA疫苗有血凝素(HA)、神经氨酸酶(NA)DNA疫苗、核壳蛋白(NP)、膜蛋白(M)DNA疫苗。联合使用细胞因子DNA对流感DNA疫苗的免疫效果有明显加强作用。众多试验表明,流感病毒DNA疫苗效果良好,其保护效果不逊于传统灭活疫苗,具有良好的发展前景。     

A型流感病毒M2蛋白疫苗的研究进展

目前用于免疫人群的流感疫苗多为三价灭活疫苗,包含A型流感病毒H1N1亚型、H3N2亚型和B型流感病毒。多年来的实践表明,三价灭活疫苗是有一定保护效果的。但是,由于流感病毒血凝素(HA)和神经氨酸酶(NA)经常发生抗原转变和抗原漂移,使其抗原性表现出很大的变异,所以根据流感疫情监测预测的疫苗株也很难产生最理想的保护效果。但流感病毒基质蛋白M2的膜外区氨基酸序列高度保守,有可能发展成为具有交叉保护能力的流感疫苗的候选抗原。该文就A型流感病毒基质蛋白M2疫苗的研究作一综述。1 A型流感病毒基质蛋白M2结构及功能流感病毒基因组RN…     

A型流感病毒血凝素、神经氨酸酶DNA疫苗研究

用BALB/C小鼠为模型,检测A型流感病毒血凝素、神经氨酸酶、基质蛋白DNA疫苗抗流感能力。研究表明:血凝素、神经氨酸酶DNA疫苗能提供有效的抗流感保护;血凝素、神经氨酸酶和基质蛋白联合免疫动物提供最佳免疫保护。     

应用单向放射免疫扩散法测定流感疫苗血凝素含量的研究

分析了单向放射免疫扩散法快速、准确测定流感疫苗血凝素含量的可行性。利用在打孔器孔径一定的条件下抗原浓度只与扩散圈直径的平方成线性关系,而与打孔器无关,测定流感疫苗血凝素含量。结果可见,流感疫苗3个型别血凝素扩散圈直径的平方均与其浓度成线性,r均大于0.99,变异系数均小于1%,平均回收率99.7%。单向放射免疫扩散法方法简便、快速、准确,适用于流感疫苗血凝素含量的测定。     

猪流感病毒血凝素基因的克隆和序列测定

本文报道了以两种化学合成的寡脱氧核苷酸引物合成了猪流感病毒NJ／11,76(X53a)株血凝素基因的全长拷贝。并将它克隆到大肠杆菌R RI细胞,进行了核苷酸全序列测定。同时将血凝素的氨基酸序列与同一亚型的另两个人甲型流感毒株的氨基酸序列进行了比较。     

用单克隆抗体选择具有潜在流行病学意义的A/曼谷79—1流感病毒变异株

用甲3型流感病毒A/曼谷79-1血凝素特异的单克隆抗体,对我国从1979—1984年分离的45株H3N2病毒进行了抗原分析,并与用单克隆抗体选择的51株A/曼谷79-1变异株作了比较。发现后者同1979年以来我国流行的H3N2流感主流株高度相似。这证明,用单克隆抗体可选出有流行潜力的变异株。本文就通过单克隆抗体选择、提前获得甲型流感亚型内变种的可能性进行了讨论。     

HPLC法测定A群C群脑膜炎球菌多糖疫苗中乳糖含量

建立了高效液相色谱法测定A群C群脑膜炎球菌多糖疫苗中乳糖的含量。样品经离心除去多糖后,采用阳离子交换柱分离,外标法定量分析。该法线性相关系数大于0.9999,回收率为98.8%,成品测定的CV值为1.2%(<2%),该法定量准确,重复性好,适于对A群C群脑膜炎球菌多糖疫苗中乳糖含量进行快速检测。     

流感病毒灭活疫苗研究进展

流感病毒的不断变异是造成流感经常流行的主要原因．研究表明人流感病毒的来源与禽流感病毒的存在密切相关．最近出现的禽流感病毒跨种属感染人的事件,预示引起下一次爆发的流感病毒流行可能直接来源于禽流感病毒．因人类对新出现的病毒缺乏免疫力,开发有效疫苗仍然是预防流感流行的关键．对流感灭活疫苗包括灭活疫苗有效成分的改良,H5N1、H9N2型人．禽流感疫苗研究和应用反向遗传技术制备流感灭活疫苗等方面的研究进展进行了探讨．     

用于快速测定流感疫苗效力的新型抗体非依赖性受体结合SPR检测法

<正>2009年H1N1流感大流行之后世界卫生组织举办研讨会,提出优先开发测定流感疫苗效力的替代检测方法,以期满足流感大流行时加快流感疫苗批签发的需求。作者开发了一种抗体非依赖性的,简单的高通量的以受体结合SPR为基础的效力检测法,这种方法不需要任何参考抗血清,而且能够用于快速血凝素定量测定以及在流感大流行情况下便于流感疫苗的快速批签发。在这种受体结合SPR表面等离振共振测定中,利用在α-2,6或者在α-2,3     

流感疫苗单向免疫扩散试验中抗血清制备新方法的研究

流感疫苗血凝素含量检测方法为单向免疫扩散试验,其抗血清通常由WHO参比实验室提供,通过纯化病毒、蛋白酶切获得血凝素主要抗原片段,然后再免疫动物获得抗血清。该方法制备时间较长,是制约流感疫苗研发及检测的主要因素。以RT-PCR方法获取甲型H1N1流感病毒血凝素中主要抗原片段基因,构建重组质粒并在大肠杆菌中表达,表达产物经纯化、复性后,以蛋白电泳及免疫印迹方法进行了鉴定。以纯化蛋白免疫家兔,制备了相应的抗体,初步证明可用于常规的单向免疫扩散试验。以该法可以快速获得抗血清,尤其是在大流行流感疫苗的研发中,加快疫苗研发进程。     

The quantification of the haemagglutinin content of influenza whole virus and Tween-ether split vaccines

Monovalent whole virus and Tween-ether split vaccines prepared from influenza A/Bangkok, A/Brazil and B/Singapore were assayed for haemagglutinin content using single radial immunodiffusion (SRID), quantitative sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunization of guinea pigs. When SRID was performed with split vaccines, haemagglutinin values were consistently recorded which were in the range of 50 to 25% of the values obtained before disruption of virions. When, however, disruption was conducted in the presence of excess detergent, thus preventing aggregate formation of solubilized haemagglutinin, test values comparable with those of whole virus vaccines were obtained. In agreement with these results, immunization experiments revealed that whole virus and corresponding split vaccines exhibited comparable immunogenicity in guinea pigs. Additionally it could be calculated from SDS-PAGE and densitometer tracings, obtained by scanning the gels after staining with either Coomassie blue or FITC-Con A, that 90 to 95% of whole virus HA2 was recovered in Tween-ether split vaccines. On the basis of these findings we conclude that precise quantification of Tween-ether split vaccines is not possible by the SRID test alone. As aggregate formation of solubilized haemagglutinin occurs, we suggest that either a physico-chemical method including a disaggregation procedure, such as SDS treatment, or immunological evaluation of the original whole virus preparation before disruption of virions should be applied as an additional criterion for quantification of influenza Tween-ether split vaccines.     

Titer on Chip: New Analytical Tool for Influenza Vaccine Potency Determination

Titer on Chip (Flu-ToC) is a new technique for quantification of influenza hemagglutinin (HA) concentration. In order to evaluate the potential of this new technique, a comparison of Flu-ToC to more conventional methods was conducted using recombinant HA produced in a baculovirus expression system as a test case. Samples from current vaccine strains were collected from four different steps in the manufacturing process. A total of 19 samples were analysed by Flu-ToC (blinded), single radial immunodiffusion (SRID), an enzyme-linked immunosorbent assay (ELISA), and the purity adjusted bicinchoninic acid assay (paBCA). The results indicated reasonable linear correlation between Flu-ToC and SRID, ELISA, and paBCA, with regression slopes of log-log plots being 0.91, 1.03, and 0.91, respectively. The average ratio for HA content measured by Flu-ToC relative to SRID, ELISA, and paBCA was 83%, 147%, and 81%, respectively; indicating nearly equivalent potency determination for Flu-ToC relative to SRID and paBCA. These results, combined with demonstrated multiplexed analysis of all components within a quadrivalent formulation and robust response to HA strains over a wide time period, support the conclusion that Flu-ToC can be used as a reliable and time-saving alternative potency assay for influenza vaccines.     

Application of deglycosylation and electrophoresis to the quantification of influenza viral hemagglutinins facilitating the production of 2009 pandemic influenza (H1N1) vaccines at multiple manufacturing sites in China

The single radial immunodiffusion (SRID) method currently used to determine the hemagglutinin (HA) content of the inactivated influenza vaccines depends on the availability of reference HA antigen and corresponding anti-serum, updated and provided annually by World Health Organization (WHO) collaborative centers. Particularly early in a pandemic outbreak, reference reagents could be the bottleneck in vaccine development and release. Therefore, other reliable tests capable of quantifying HA content could substantially shorten the time needed for vaccine formulation. Here electrophoretic separation of deglycosylated samples in conjunction with densitometry was used to quantify HA contents of H1N1 vaccine at multiple manufacturing sites. We found the overall consistency between the alternative method and traditional SRID was 88–122% in seven lots of vaccine bulks from four subtypes (types) of influenza vaccine, confirming its suitability to quantify HA content. Moreover, we used the alternative method to prepare a national HA antigen reference in China for quality control of 2009 pandemic influenza A (H1N1) vaccines prior to the arrival of the WHO SRID reference standards, subsequently confirming good agreement between both methods. The alternative method for vaccine quantification enabled the Chinese health authority to approve H1N1 vaccine 1 month earlier than otherwise possible.     

Origin and evolution of influenza virus hemagglutinin genes

Influenza A, B, and C viruses are the etiological agents of influenza. Hemagglutinin (HA) is the major envelope glycoprotein of influenza A and B viruses, and hemagglutinin-esterase (HE) in influenza C viruses is a protein homologous to HA. Because influenza A virus pandemics in humans appear to occur when new subtypes of HA genes are introduced from aquatic birds that are known to be the natural reservoir of the viruses, an understanding of the origin and evolution of HA genes is of particular importance. We therefore conducted a phylogenetic analysis of HA and HE genes and showed that the influenza A and B virus HA genes diverged much earlier than the divergence between different subtypes of influenza A virus HA genes. The rate of amino acid substitution for A virus HAs from duck, a natural reservoir, was estimated to be 3.19 x 10(-4) per site per year, which was slower than that for human and swine A virus HAs but similar to that for influenza B and C virus HAs (HEs). Using this substitution rate from the duck, we estimated that the divergences between different subtypes of A virus HA genes occurred from several thousand to several hundred years ago. In particular, the earliest divergence time was estimated to be about 2,000 years ago. Also, the A virus HA gene diverged from the B virus HA gene about 4,000 years ago and from the C virus HE gene about 8,000 years ago. These time estimates are much earlier than the previous ones.     

A Novel Activation Mechanism of Avian Influenza Virus H9N2 by Furin

Avian influenza virus H9N2 is prevalent in waterfowl and has become endemic in poultry in Asia and the Middle East. H9N2 influenza viruses have served as a reservoir of internal genes for other avian influenza viruses that infect humans, and several cases of human infection by H9N2 influenza viruses have indicated its pandemic potential. Fortunately, an extensive surveillance program enables close monitoring of H9N2 influenza viruses worldwide and has generated a large repository of virus sequences and phylogenetic information. Despite the large quantity of sequences in different databases, very little is known about specific virus isolates and their pathogenesis. Here, we characterize a low-pathogenicity avian influenza virus, A/chicken/Israel/810/2001 (H9N2) (Israel810), which is representative of influenza virus strains that have caused severe morbidity and mortality in poultry farms. We show that under certain circumstances the Israel810 hemagglutinin (HA) can be activated by furin, a hallmark of highly pathogenic avian influenza virus. We demonstrate that Israel810 HA can be cleaved in cells with high levels of furin expression and that a mutation that eliminates a glycosylation site in HA₁ allows the Israel810 HA to gain universal cleavage in cell culture. Pseudoparticles generated from Israel810 HA, or the glycosylation mutant, transduce cells efficiently. In contrast, introduction of a polybasic cleavage site into Israel810 HA leads to pseudoviruses that are compromised for transduction. Our data indicate a mechanism for an H9N2 evolutionary pathway that may allow it to gain virulence in a distinct manner from H5 and H7 influenza viruses.     

Mutations at palmitylation sites of the influenza virus hemagglutinin affect virus formation.

The carboxy terminus of the hemagglutinin (HA) of influenza A viruses contains three cysteine residues which are highly conserved among HA subtypes. It has previously been shown for the H2, H3, and H7 subtypes of HA that these cysteine residues are modified by the covalent attachment of palmitic acid. In order to study the role of the acylated cysteines in the formation of infectious influenza viruses, we introduced mutations into the HA of influenza A/WSN/33 virus (H1 subtype) by reverse-genetics techniques. We found that the cysteine at position 563 of the cytoplasmic tail is required for infectious-particle formation. The cysteine at position 560 can be changed to alanine or tyrosine to yield virus strains that are attenuated in cell cultures. The change from cysteine at position 553 to serine or alanine does not significantly alter the phenotype of the virus. The requirement for a cysteine at position 563 suggests a functional role for palmitylation of the cytoplasmic tail. This interpretation is further supported by experiments in which two or more of the cysteine residues were mutated, eliminating potential palmitylation sites. None of these double or triple mutations resulted in infectious virus. Selection of revertants of the attenuated cysteine-to-tyrosine mutant (mutation at position 560) always resulted in reversion to cysteine rather than to other amino acids. Although our data indicate a biological role for the conserved cysteine residues in the cytoplasmic tail of the HA of influenza viruses, we cannot exclude the possibility that structural constraints in the cytoplasmic tail of the HA--rather than altered palmitylation--are the determining factors for infectious-particle formation.     

一种含不同流感病毒血凝素抗原表位的核酸疫苗

流感病毒表面抗原血凝素( hemagglutinin,HA)是流感核酸疫苗重要的靶抗原,针对HA的保护性中和抗体主要由HA上的五个抗原表位诱导产生.在本文中,我们构建了一种以新甲型H1N1流感病毒HA1为骨架的含2个A/PR/8( H1N1)流感病毒HA抗原表位和3个新甲型H1N1流感病毒HA抗原表位的核酸疫苗,并在B...     

Application of recombinant hemagglutinin proteins as alternative antigen standards for pandemic influenza vaccines

Objectives

The single radial immunodiffusion (SRID) assay, used to quantify hemagglutinin (HA) in influenza vaccines, requires reference reagents; however, because centralized production of reference reagents may slow the emergency deployment of vaccines, alternatives are needed.

Results

We investigated the production of HA proteins using recombinant DNA technology, rather than a traditional egg-based production process. The HA proteins were then used in an SRID assay as a reference antigen. We found that HA can be quantified in both egg-based and cell-based influenza vaccines when recombinant HAs (rHAs) are used as the reference antigen. Furthermore, we confirmed that rHAs obtained from strains with pandemic potential, such as H5N1, H7N3, H7N9, and H9N2 strains, can be utilized in the SRID assay. The rHA production process takes just one month, in contrast to the traditional process that takes three to four months.

Conclusions

The use of rHAs may reduce the time required to produce reference reagents and facilitate timely introduction of vaccines during emergencies.

     

Annexin II incorporated into influenza virus particles supports virus replication by converting plasminogen into plasmin

For influenza viruses to become infectious, the proteolytic cleavage of hemagglutinin (HA) is essential. This usually is mediated by trypsin-like proteases in the respiratory tract. The binding of plasminogen to influenza virus A/WSN/33 leads to the cleavage of HA, a feature determining its pathogenicity and neurotropism in mice. Here, we demonstrate that plasminogen also promotes the replication of other influenza virus strains. The inhibition of the conversion of plasminogen into plasmin blocked influenza virus replication. Evidence is provided that the activation of plasminogen is mediated by the host cellular protein annexin II, which is incorporated into the virus particles. Indeed, the inhibition of plasminogen binding to annexin II by using a competitive inhibitor inhibits plasminogen activation into plasmin. Collectively, these results indicate that the annexin II-mediated activation of plasminogen supports the replication of influenza viruses, which may contribute to their pathogenicity.