B型流感病毒血凝素的表达及免疫原性测定北大核心CSCD

B型流感病毒是引起季节性流感的原因之一,严重时会造成重大疾病或死亡。为了检测B型流感病毒2个疫苗候选毒株的血凝素(hemagglutinin,HA)蛋白胞外段在哺乳动物细胞中的表达及在小鼠体内的免疫原性,本研究将带有三聚体标签的HA胞外段(HA-ectodomain,HA-ecto)序列及神经氨酸酶(neuraminidase,NA)全长编码框经密码子优化后构建至pCAGGS载体中,通过线性聚乙烯亚胺将pCAGGS-HA-ecto与pCAGGS-NA共转染293T细胞。收集转染后96h的上清,通过镍离子亲和层析及分子筛层析获得三聚体形式的HA-ecto蛋白,然后将HA-ecto三聚体蛋白免疫小鼠,进行酶联免疫吸附试验(enzyme linked immunosorbent assay,ELISA)及血凝抑制实验(hemagglutination inhibition,HAI)检测HA-ecto蛋白诱导小鼠后产生的抗体水平。纯化结果显示,通过哺乳动物细胞表达系统能够得到分泌型表达的三聚体HA-ecto蛋白。ELISA及HAI结果显示,三聚体HA-ecto蛋白二次免疫小鼠后,能诱导小鼠产生较高水平的同源和异源交叉抗体。以上结果表明,哺乳动物细胞表达的B型流感病毒HA蛋白可作为亚单位重组流感疫苗的候选。     

Experimental evolution of human influenza virus H3 hemagglutinin in the mouse lung identifies adaptive regions in HA1 and HA2

The genetic basis for virulence and host switching in influenza A viruses (FLUAV) is largely unknown. Because the hemagglutinin (HA) protein is a determinant of these properties, HA evolution was mapped in an experimental model of mouse lung adaptation. Variants of prototype A/Hong Kong/1/68 (H3N2) (wild-type [wt] HK) human virus were selected in both longitudinal and parallel studies of lung adaptation. Mapping of HA mutations found in 11 independently derived mouse-adapted populations of wt HK identified 27 mutations that clustered within two distinct regions in or near the globular frameworks of the HA1 and HA2 subunits. The adaptive mutations demonstrated multiple instances of convergent evolution involving four amino acid positions (162, 210, and 218 in HA1 and 154 in HA2). By use of reverse genetics, convergent HA mutations were shown to affect cell tropism by enhancing infection and replication in primary mouse tracheal epithelial cells in vitro and mouse lung tissue in vivo. Adaptive HA mutations were multifunctional, affecting both median pH of fusion and receptor specificity. Specific mutations within both adaptive regions were shown to increase virulence in a mouse lung model. The occurrence of mutations in the HA1 and HA2 adaptive regions of natural FLUAV host range and virulent variants of avian and mammalian viruses is discussed. This study has identified adaptive sites and regions within the HA1 and HA2 subunits that may guide future studies of viral adaptation and evolution in nature.     

Directed reconstruction of the influenza virus hemagglutinin gene

A new approach to create chimeric genes by directed exchange of oligonucleotide fragments was developed. By oligonucleotide-directed mutagenesis a few deletion mutants of the influenza virus hemagglutinin (HA) gene were obtained. These variants of HA gene contain unique restriction sites in DNA regions coding for the A and B epitopes of the HA molecule. The obtained special vectors may be used for cloning DNA fragments coding for new amino acid sequences in internal sites of the HA gene.     

Glycosylation focuses sequence variation in the influenza A virus H1 hemagglutinin globular domain

Antigenic drift in the influenza A virus hemagglutinin (HA) is responsible for seasonal reformulation of influenza vaccines. Here, we address an important and largely overlooked issue in antigenic drift: how does the number and location of glycosylation sites affect HA evolution in man? We analyzed the glycosylation status of all full-length H1 subtype HA sequences available in the NCBI influenza database. We devised the “flow index” (FI), a simple algorithm that calculates the tendency for viruses to gain or lose consensus glycosylation sites. The FI predicts the predominance of glycosylation states among existing strains. Our analyses show that while the number of glycosylation sites in the HA globular domain does not influence the overall magnitude of variation in defined antigenic regions, variation focuses on those regions unshielded by glycosylation. This supports the conclusion that glycosylation generally shields HA from antibody-mediated neutralization, and implies that fitness costs in accommodating oligosaccharides limit virus escape via HA hyperglycosylation.     

The 1-127 HA2 construct of influenza virus hemagglutinin induces cell-cell hemifusion.

Conformational changes in the HA2 subunit of influenza hemagglutinin (HA) are coupled to membrane fusion. We investigated the fusogenic activity of the polypeptide FHA2 representing 127 amino-terminal residues of the ectodomain of HA2. While the conformation of FHA2 both at neutral and at low pH is nearly identical to the final low-pH conformation of HA2, FHA2 still induces lipid mixing between liposomes in a low-pH-dependent manner. Here, we found that FHA2 induces lipid mixing between bound cells, indicating that the "spring-loaded" energy is not required for FHA2-mediated membrane merger. Although, unlike HA, FHA2 did not form an expanding fusion pore, both acidic pH and membrane concentrations of FHA2, required for lipid mixing, have been close to those required for HA-mediated fusion. Similar to what is observed for HA, FHA2-induced lipid mixing was reversibly blocked by lysophosphatidylcholine and low temperature, 4 degrees C. The same genetic modification of the fusion peptide inhibits both HA- and FHA2-fusogenic activities. The kink region of FHA2, critical for FHA2-mediated lipid mixing, was exposed in the low-pH conformation of the whole HA prior to fusion. The ability of FHA2 to mediate lipid mixing very similar to HA-mediated lipid mixing is consistent with the hypothesis that hemifusion requires just a portion of the energy released in the conformational change of HA at acidic pH.     

Sublingual administration of bacteria-expressed influenza virus hemagglutinin 1 (HA1) induces protection against infection with 2009 pandemic H1N1 influenza virus

Influenza viruses are respiratory pathogens that continue to pose a significantly high risk of morbidity and mortality of humans worldwide. Vaccination is one of the most effective strategies for minimizing damages by influenza outbreaks. In addition, rapid development and production of efficient vaccine with convenient administration is required in case of influenza pandemic. In this study, we generated recombinant influenza virus hemagglutinin protein 1 (sHA1) of 2009 pandemic influenza virus as a vaccine candidate using a well-established bacterial expression system and administered it into mice via sublingual (s.l.) route. We found that s.l. immunization with the recombinant sHA1 plus cholera toxin (CT) induced mucosal antibodies as well as systemic antibodies including neutralizing Abs and provided complete protection against infection with pandemic influenza virus A/CA/04/09 (H1N1) in mice. Indeed, the protection efficacy was comparable with that induced by intramuscular (i.m.) immunization route utilized as general administration route of influenza vaccine. These results suggest that s.l. vaccination with the recombinant non-glycosylated HA1 protein offers an alternative strategy to control influenza outbreaks including pandemics.     

H5N1亚型禽流感病毒HA基因核酸序列的变异分析

从GenBank上获得194株不同来源的H5N1亚型禽流感病毒HA基因核酸序列,利用MEGA3分析了HA基N核酸序列碱基突变的特点。并通过比较序列同源性,构建NJ系统进化树,探讨了不同来源的H5N1病毒的系统进化关系。序列分析结果表明,被研究的194条H5N1亚型禽流感病毒HA基因核酸序列,碱基长度大约在1700bp左右,共发现了757个可变位点,其中Parsimony—informative sites有537个,Singleton sites有220个;病毒的变异速率很快,平均变异率为3．23％;病毒的序列变异具有显著的地区特点和时间特点;同时,全球化的贸易以及候鸟的迁徙在传播病毒过程中起一定作用。     

Protonation and stability of the globular domain of influenza virus hemagglutinin.

A partial dissociation of the HA1 subunits of influenza virus hemagglutinin (HA) is considered to be the initial step of conformational changes of the HA ectodomain leading to a membrane fusion active conformation (L. Godley, J. Pfeifer, D. Steinhauer, B. Ely, G. Shaw, R. Kaufman, E. Suchanek, C. Pabo, J.J. Skehel, D.C. Wiley, and S. Wharton, 1992, Cell 68:635-645; G.W. Kemble, D.L.Bodian, J. Rose, I.A. Wilson, and J.M. White, 1992, J. Virol. 66:4940-4950). Here, we explore a mechanism that provides an understanding of the physical and chemical basis for such dissociation and relies on two essential observations. First, based on the x-ray structure of HA from X31 (I.A. Wilson, J.J. Skehel, and D.C. Wiley, 1981, Nature 289:366-373), and by employing techniques of molecular modeling, we show that the protonation of the HA1 subunits is enhanced at the conditions known to trigger conformational changes of the HA ectodomain. Second, we found that the dependence of the calculated relative degree of protonation of the HA1 domain on temperature and pH is similar to that observed experimentally for the conformational change of HA assessed by proteinase K sensitivity. We suggest that at the pH-temperature conditions typical for the conformational change of HA and membrane fusion, dissociation of the HA1 subunits is caused by the enhanced protonation of the HA1 subunits leading to an increase in the positive net charge of these subunits and, in turn, to a weakened attraction between them.     

Isolation of influenza virus A hemagglutinin C-terminal domain by hemagglutinin proteolysis in octylglucoside micelles

A method of isolation of hydrophobic membrane-bound C-terminal domain of influenza virus A hemagglutinin (HA) is suggested. The method is based on the insertion of HA into octylglucoside micelles followed by pepsin or thermolysin hydrolysis. Subsequent treatment of proteolytic digests with chloroform-hexafluoroisopropanol mixture resulted in the extraction of a few hydrophobic peptides into organic phase. Mass-spectrometry (MALDI-TOF) analysis revealed that the peptides with ion masses corresponding to the anchoring C-terminal domain with or without modifications predominated in the organic solution. The data obtained confirmed our speculation on the possibility of the suggested isolation scheme following from the strong interactions of anchoring domains in compact trimeric structure of HA spikes combined with micelle protection effect. Several appropriate peptides presence in the organic phase apparently arises from the presence of a few accessible proteolytic sites in HA transmembrane region.     

Structure of the hemagglutinin gene of the influenza virus during serial passages in chick embryos

The sequence analysis of HA gene of passaged variants of A/USSR/2/85 influenza virus was carried out. It was shown that in the process of passage of virus in chick embryos the structure of HA gene was not changed. These data correlated with those obtained early during investigation of A/Leningrad/337/76 influenza virus by the oligonucleotide mapping.     

Recognition of influenza virus hemagglutinin by subtype-specific and cross-reactive proliferative T cells: contribution of HA1 and HA2 polypeptide chains

The recognition of influenza virus hemagglutinin (HA) by T lymphocytes was examined by assaying the T cell proliferative response of influenza virus-primed T cells to purified HA of different influenza A subtypes or to isolated heavy (HA1) or light (HA2) polypeptide chains of the HA molecule. The proliferative response to HA was dependent on the activation of an Ly-1+2- subset of T cells and required the presence of nylon wool-adherent, radiation-resistant accessory cells. T cells from mice primed by infection with one strain of type A influenza virus cross-reacted with other purified HA not only of the same subtype as the priming virus but also of serologically distinct subtypes of influenza A (but not B) virus. The response of virus-primed T cells to the homologous HA or to HA of the same subtype was shown to involve recognition of determinants on both the HA1 and the HA2 chains. The recognition of HA of different subtype by cross-reactive T cells appeared to be directed predominantly to determinants on HA2. Because the antibody response to influenza virus HA is not cross-reactive between subtypes and is directed predominantly to determinants on HA1, the present results indicate that at least some of the determinants on HA recognized by T cells are different from those recognized by B cells and that the HA2 chain may be involved primarily in stimulation of T cell rather than B cell immunity.     

The receptor-binding domain of influenza virus hemagglutinin produced in Escherichia coli folds into its native, immunogenic structure

The hemagglutinin (HA) surface glycoprotein promotes influenza virus entry and is the key protective antigen in natural immunity and vaccines. The HA protein is a trimeric envelope glycoprotein consisting of a globular receptor-binding domain (HA-RBD) that is inserted into a membrane fusion-mediating stalk domain. Similar to other class I viral fusion proteins, the fusogenic stalk domain spontaneously refolds into its postfusion conformation when expressed in isolation, consistent with this domain being trapped in a metastable conformation. Using X-ray crystallography, we show that the influenza virus HA-RBD refolds spontaneously into its native, immunogenic structure even when expressed in an unglycosylated form in Escherichia coli. In the 2.10-Å structure of the HA-RBD, the receptor-binding pocket is intact and its conformational epitopes are preserved. Recombinant HA-RBD is immunogenic and protective in ferrets, and the protein also binds with specificity to sera from influenza virus-infected humans. Overall, the data provide a structural basis for the rapid production of influenza vaccines in E. coli. From an evolutionary standpoint, the ability of the HA-RBD to refold spontaneously into its native conformation suggests that influenza virus acquired this domain as an insertion into an ancestral membrane-fusion domain. The insertion of independently folding domains into fusogenic stalk domains may be a common feature of class I viral fusion proteins.The genetic drift of seasonal influenza viruses and the occasional emergence of pandemic strains represent a continuing and serious burden on human health. Pandemic influenza viruses arise at irregular intervals, can infect up to 50% or more of the population, and vary in disease severity. Most notably, the H1N1 Spanish influenza pandemic of 1918 killed an estimated 20 to 50 million people worldwide, and the 1957 H2N2 Asian flu and 1968 H3N2 Hong Kong flu pandemics killed between 0.5 and 1 million people in the United States alone (30). The ongoing danger of influenza was recently emphasized by the emergence of the novel H1N1 pandemic virus from Mexico in April of 2009. The urgent need to speed up vaccine production was highlighted by this outbreak because over 340,000 confirmed cases and 4,100 deaths had occurred worldwide during the 6 months that were necessary to produce a vaccine using current procedures (39).As the major surface antigen of influenza A viruses, the hemagglutinin (HA) envelope glycoprotein is the primary source of natural immunity and the key target in vaccination. However, changes in the antigenic sites of the HA protein due to antigenic drift result in lost or diminished immunity acquired from previous infection or vaccination (35). This necessitates the production of new vaccines against seasonal influenza viruses each year. The HA protein also plays a central role in the emergence of human pandemic influenza viruses. There are 16 known antigenic subtypes of HA proteins in influenza A viruses (H1 through H16), and a pandemic occurs when an influenza virus that has an HA protein to which most of the population lacks immunity acquires the ability to be efficiently transmitted from person to person.The HA protein has multiple roles in the virus life cycle, notably receptor binding and membrane fusion. The protein is synthesized as a single precursor protein, HA₀, that trimerizes and becomes glycosylated in the endoplasmic reticulum as it traffics to the cell surface (33). The HA protein contains multiple disulfide bonds and is cleaved into a mature form consisting of two subunits, HA₁ and HA₂ (9, 18). HA₂ and the N- and C-terminal portions of HA₁ form a membrane-proximal stalk that mediates membrane fusion during viral entry (40). A receptor-binding domain (HA-RBD) forms the distal head of the molecule and is inserted into the HA₁ subunit. During virus entry, the HA-RBD engages sialic acid-containing receptors on the surface of the host cell, and the virion is subsequently internalized by endocytosis (33). Structurally and functionally, the HA-RBD is a member of the lectin superfamily, and the specificity of the binding pocket contributes to the host range of influenza viruses. For example, α(2,6)-containing sialosides are typically preferred by the HA protein from human viruses and α(2,3) sialosides by the HA proteins from avian viruses (13, 28). Upon triggering by the low-pH environment of endosomes, the HA protein undergoes an irreversible conformational change (6, 40) during which the intact HA-RBDs dissociate from the stalk of the trimer (3, 14, 19, 21). This observation, together with the manner in which the lectin-like domain is inserted as a folded module into the full-length HA protein, led us to hypothesize that the HA-RBD is able to adopt its native structure in isolation. Proper folding of the isolated HA-RBD into its native immunogenic structure has important therapeutic implications because the domain contains all of the known HA antigenic epitopes responsible for antibody recognition (5), and producing a protein-based influenza vaccine composed of isolated HA-RBD would dramatically speed up vaccine development during the early stages of a pandemic.In a recently published report, a construct of the 2009 pandemic H1N1 HA protein that encompasses the HA-RBD, designated HA_63-286-RBD, was expressed in Escherichia coli as inclusion bodies, refolded and purified, and used as a vaccine to produce immunity in ferrets (2). In this report, we show that this construct behaves as a stable, structured protein in solution, can be readily crystallized, and indeed adopts a structure that is virtually indistinguishable from that in the H1N1 HA protein ectodomain (41).     

辽宁省2016‒2017年H3N2亚型流感病毒HA1基因特征分析

目的 了解2016‒2017年辽宁省H3N2亚型流感病毒基因变异情况及流行株与疫苗株的匹配情况。方法 采用逆转录聚合酶链反应（RT-PCR）对分离得到的H3N2亚型流感毒株的HA1基因进行扩增,扩增片段经测序与近年来WHO推荐的北半球疫苗株进行比对和基因特征分析。结果 进化分析表明,2016‒2017年H3N2亚型流感病毒与近三年的疫苗株均不在同一分支上;基因特性分析中,所有病毒均在A、B抗原决定簇上发生了两处以上的变异;19株病毒的受体结合位点131位氨基酸发生了新的变异;20株病毒中有1株突变产生了新的半胱氨酸,提示可能有新的二硫键产生;糖基化位点并未检测到新的突变。结论 2016‒2017年辽宁省H3N2亚型流感病毒的抗原性及基因特性均发生了一定的变化,但变异程度不大,应密切关注疫苗株对流感病毒的免疫效果及流感毒株的变异情况。     

Antigenic determinants of influenza virus hemagglutinin. V. Antigenicity of the HA2 chain

All the polypeptide fragments obtained by cyanogen bromide cleavage of the hemagglutinin from A/Memphis/102/72 influenza virus were examined for their ability to bind to IgG raised against purified virus. Within the hemagglutinin heavy chain the only fragment displaying antigenicity is HA1CN1, which comprises the amino-terminal 168 amino acid residues. By the use of a sensitive radioimmunoassay in which the antigen is unlabeled, it was shown that the light chain is also antigenic. Inhibition studies have localized the activity to the HA2CN1 region, which comprises the carboxy-terminal 90 amino acids. The determinant on HA2 is shown to be subtype specific.     

Cloning and expression of the hemagglutinin gene of influenza virus subtype H1 in Escherichia coli

The full-length copy of the hemagglutinin gene of influenza virus was inserted into M13 phage DNA. The DNA sequence coding for the hydrophobic prepeptide was removed from the gene by oligonucleotide-directed mutagenesis. The possibilities of expression of the full-length and mutant genes in E. coli were investigated. The beta-galactosidase-hemagglutinin fusion proteins were isolated. The fusion proteins exhibited specific binding to antiviral antibodies. This binding could be competitively inhibited by excess of viral hemagglutinin, demonstrating that these fusion proteins contained antigenic determinants of hemagglutinin.     

Disulfide bond formation during the folding of influenza virus hemagglutinin

To study the importance of individual sulfhydryl residues during the folding and assembly in vivo of influenza virus hemagglutinin (HA), we have constructed and expressed a series of mutant HA proteins in which cysteines involved in three disulfide bonds have been substituted by serine residues. Investigations of the structure and intracellular transport of the mutant proteins indicate that (a) cysteine residues in the ectodomain are essential both for efficient folding of HA and for stabilization of the folded molecule; (b) cysteine residues in the globular portion of the ectodomain are likely to form native disulfide bonds rapidly and directly, without involvement of intermediate, nonnative linkages; and (c) cysteine residues in the stalk portion of the ectodomain also appear not to form intermediate disulfide bonds, even though they have the opportunity to do so, being separated from their correct partners by hundreds of amino acids including two or more other sulfhydryl residues. We propose a role for the cellular protein BiP in shielding the cysteine residues of the stalk domain during the folding process, thus preventing them from forming intermediate, nonnative disulfide bonds.     

Evolutionary pattern of the hemagglutinin gene of influenza B viruses isolated in Japan: cocirculating lineages in the same epidemic season.

The unexpectedly low efficacy of influenza vaccine during school outbreaks of influenza B virus in the spring of 1987 in Japan was probably attributable to a poor antibody response of vaccinees to the epidemic viruses. An antigenic analysis of the causative B viruses isolated in 1987 and 1988 showed much variation in hemagglutination inhibition patterns. The nucleotide sequences that code for the HA1 domain of B/Fukuoka/c-27/81, B/Ibaraki/2/85, B/Nagasaki/1/87, and B/Yamagata/16/88 viruses were determined and compared with those of the previously reported hemagglutinin genes. The nucleotide sequences of the hemagglutinin gene of a new variant, B/Yamagata/16/88, had only 93.4% homology with those of two other viruses from the same epidemic. An analysis of nucleotide and amino acid substitutions of the hemagglutinin genes of influenza B viruses revealed that new and some old variants could cocirculate in the same epidemic. A phylogenetic tree constructed by the neighbor-joining method allowed estimation of an evolutionary rate of 2.3 x 10(-3) synonymous (silent) substitutions per nucleotide site per year in the hemagglutinin gene.     

一株2011年出现的季节性甲型H1N1流行性感冒病毒血凝素基因特性的研究

本文通过比较2011年分离培养的1株季节性甲型H1N1流行性感冒(简称流感)病毒(A/Shanghai/1167/2011(H1N1))与历年季节性甲型H1N1流感病毒的血凝素(HA)基因,追溯该病毒的基因变异与来源,探讨该毒株的出现对流感防控工作的意义.采用反转录-聚合酶链反应(RT-PCR)方法扩增病毒的HA和神经氨酸酶(NA)片段,并进行测序;应用分子生物学软件对获得的序列进行分析,绘制基因进化树;同时,通过血凝抑制试验检测2011年下半年健康人群中该流感病毒的抗体水平.结果显示,A/Shanghai/1167/2011(H1N1)的HA基因序列与世界卫生组织(WHO)2007～2008年季节性甲型H1N1流感病毒疫苗株A/Brisbane/59/2007(H1N1)最接近,同源性达99.2%,与新型甲型H1N1流感病毒A/California/07/2009疫苗株同源性仅为72.4%.其HA基因裂解位点为PSIQSR↓GLF,尚未出现高致病性的分子特征.HA片段共编码557个氨基酸,有9个潜在的糖基化位点,序列与2009年前WHO疫苗株A/NewCaledonia/20/1999(H1N1)、A/SolomonIslands/3/2006(H1N1)和/Brisbane/59/2007(H1N1)相比,分别有15、12和4处不同,这些差异分布在Sa、Sb、Ca1、Ca2、Cb 5个抗原决定簇的氨基酸差异分别有5、5和2处.该毒株在健康人群血清的抗体阳性率为34.33%,几何平均效价(GMT)为10.38.A/Shanghai/1167/2011(H1N1)是2011年出现在上海地区的一个季节性甲型H1N1流感病毒毒株,其抗原变异与既往季节性甲型H1N1流感病毒相比不大,但在以A(H1N1)pdm09为主要流行株的年份检测到散在发生的既往季节性甲型H1N1流感病毒毒株应当引起重视,其在人群中的抗体水平较低,易引起流行,需要提高对类流感人群中此种毒株的持续监测.