Purification of haemagglutinin and neuraminidase from influenza virus strain 3QB and isolation of a peptide from an antigenic region of haemagglutinin

Methods were developed for the purification of the surface, membrane-bound glycoproteins haemagglutinin and neuraminidase of influenza virus strain 3QB, in antigenically active forms. The methods employed in the purification included selective removal of the neuraminidase with the proteinase, bromelain, and subsequent disruption of the residual virus particle with the detergent Sarkosyl to release the haemagglutinin. Using techniques for proteolytic digestion of intact, native proteins an antigenically active peptide was isolated from the purified haemagglutinin, the surface glycoprotein against which the major antigenic response is directed. The amino acid composition of this peptide was determined. This was a 16-residue peptide with amino-terminal isoleucine and composition Ile1 Val1 Asx2 Thr1 Ser2 Glx2 Pro1 Gly3 Ala1 Leu1 Lys1.     

Hemagglutinin specificity of Cl. botulinum types A, B, and F in reaction with erythrocytes of various animals]

The following differences were revealed in the haemagglutination reaction with the erythrocytes of man, sheep, rabbit, chicks and mice between the haemagglutinins of Cl. botulinum, types A, B and F, having a close affinity with one another: haemagglutinin of type A actively reacted with the erythrocytes of man, sheep, rabbit, rats and chicks; haemagglutinin of type B reacted only with the erythrocytes of man and rabbits; haemagglutinin of type F failed to react with any of the types of the erythrocytes tested; only with the use of erythrocytes treated with neuraminidase was it possible to establish the presence of haemagglutinin fraction in Cl. botulinum, type F. Treatment of human erythrocytes with neuraminidase and proteolytic enzymes caused a marked increase in the sensitivity of the haemagglutination reaction in haemagglutinins of Cl. botulinum, types A, B and F.     

Intracellular processing and transport of influenza-virus envelope proteins in Madin-Darby canine kidney cells. Effects of the carboxylic ionophores monensin and nigericin.

We have investigated the effects of the carboxylic ionophores monensin and nigericin on the intracellular processing and transport of the influenza-virus envelope proteins haemagglutinin and neuraminidase in Madin-Darby-canine-kidney-cell monolayers. In the presence of either ionophore, haemagglutinin acquires resistance to the enzyme endoglycosidase H more slowly than it does in untreated cells. In addition, the ionophores cause a block in oligosaccharide-processing events that are believed to occur normally in the trans elements of the Golgi complex. This block is not overcome even at long chase times. Finally, the ionophores cause a substantial slowing of the delivery of both haemagglutinin and neuraminidase to the plasma membrane. We conclude that the ionophores cause delays in the intracellular transport of these proteins both early and late in the pathway, that is, before and after passage through the trans-Golgi, and perturb the processing functions of this compartment. The possible significance of these observations with regard to the intracellular transport of newly synthesized plasma-membrane proteins in epithelial cells is discussed.     

Crystalline Antigen from the Influenza Virus Envelope

THE envelope of influenza viruses contains two morphologically and chemically distinct glycoproteins, a haemagglu-tinin and a neuraminidase, which recent experiments have indicated¹ may be removed from the virion by treatment with the protease, bromelain. In an investigation of the mechanism of this phenomenon it was found that although the neuraminidase protein was completely degraded during proteolysis, the isolated haemagglutinin protein could be recovered almost unchanged from the incubation mixture. We report the subsequent purification and crystallization of the haemagglutinin and its preliminary chemical and antigenic characterization. The X-31 strain of A2/Hong Kong/68² was used in most of the experiments to be described. Viruses were grown in embryonated eggs, purified as described by Skehel and Schild³ and digested with bromelain as described by Compans et al.¹. The smooth-surfaced virus particles which resulted were removed from the incubation mixture by centrifuging for 60 min at 100,000g and purified by sucrose density gradient centrifugation (20–60% sucrose in phosphate buffered saline (PBS) (100,000g, 16 h). The morphology of these particles in comparison with intact virus is shown in Fig. 1. It is clear that all of the envelope spike proteins were removed during proteolytic digestion; in agreement with these observations the shaved particles were completely devoid of the haemagglutinating and neuraminidase activities of the virus envelope proteins. Polyacrylamide gel electrophoretic analyses of the polypeptide composition of the particles also indicated that they contained only four of the seven polypeptides of the virus⁴ in the same proportions as these were present in the intact virus particles (Fig. 2b and c). Both haemagglu-tination and neuraminidase activities were completely destroyed during protease treatment. Electrophoretic analyses of the proteins present in the protease incubation supernatant after removal of the subviral particles indicated, however (Fig. 2a), that the major components of this mixture were two polypeptides of similar mobility to the haemagglutinin polypeptides previously identified³. Thus, the mobility of the larger polypeptide of the supernatant was identical to that of the larger haemagglutinin component and the mobility of the smaller polypeptide indicated a molecular weight difference of approximately 3,000 between this and the smaller haemagglutinin polypeptide.     

An overview of influenza haemagglutinin and neuraminidase

This is an overview of the structures of influenza virus haemagglutinin and neuraminidase membrane glycoproteins with particular reference to antibody recognition and antigenic variation.     

Evaluation of antibody response in mice against avian influenza A (H5N1) strain neuraminidase expressed in yeast Pichia pastoris

Avian influenza has raised many apprehension in the recent years because of its potential transmitability to humans. With the increasing emergence of drug-resistant avian influenza strains, development of potential vaccines are imperative to manage this disease. Two structural antigens, haemagglutinin and neuraminidase, have been the target candidates for the development of subunit vaccine against influenza. In an effort to develop a faster and economically beneficial vaccine, the neuraminidase gene of a highly pathogenic avian influenza isolate was cloned and expressed in the methylotrophic yeast Pichia pastoris. The recombinant neuraminidase (rNA) antigen was purified, and its bioactivity was analysed. The rNA was found to be functional, as determined by the neuraminidase assay. Four groups of mice were immunized with different concentrations of purified rNA antigen, which were adjuvanted with aluminium hydroxide. The immune response against rNA was analysed by enzyme-linked immunosorbent assay (ELISA) and neuraminidase inhibition assay. The mice groups immunized with 25 μg and 10 μg of antigen had a significant immune response against rNA. This method can be utilized for faster and cost-effective development of vaccines for a circulating and newer strain of avian influenza, and would aid in combating the disease in a pandemic situation, in which production time matters greatly.     

Antigenic relationship of influenza-virus neuraminidases from Asian,Hong Kong,and Equi-2 strains

Two Asian influenza strains, the Hong Kong strain and an Equi-2 influenza strain have been investigated. Adsorption isotherms of purified neuraminidases from these strains with isolated antibodies from homologous antisera are given. Cross testing by neuraminidase inhibition and haemagglutination inhibition are reported. Hong Kong neuraminidase appears to be indistinguishable from the earlier Asian neuraminidases, and not related to Equi-2 influenza neuraminidase. The haemagglutinin from the Hong Kong strain is related to earlier Asian strains as well as to the equine strain.In collaboration with the Chief Medical Officer of Health and Epidemiology, Department of Public Health, Den Haag, The Netherlands.It is a pleasure to express our thanks to Mr. J. Jacobs, N. V. Philips Duphar, Weesp (The Netherlands) for his interesting discussions and suggestions; and to Mr. H. de Jonge, Department of Medical Statistics, University of Leiden (The Netherlands) for his advice.     

Treatment of influenza with neuraminidase inhibitors: virological implications.

Evaluation of the emergence of influenza virus resistance to neuraminidase inhibitors (NAIs) is now demanded following experience with amantadinamines. Preliminary data have indicated that NAI-resistant virus is unlikely to emerge readily in the clinic and this is consistent with the difficulty experienced in selecting resistant virus in vitro. Resistance mutations can occur in both neuraminidase and haemagglutinin genes. The neuraminidase mutations are viral subtype specific and, therefore, clinically relevant subtypes must be employed for in vitro studies if pre-clinical data are to have predictive value. Haemagglutinin mutations generated in vitro are probably both subtype and cell culture system specific and, therefore, may not be predictive of clinical findings. Analysis of influenza-positive samples from NAI-treated patients in the clinical setting must include samples from late treatment time-points (day 4 and later) in order for resistant virus to be detected as in vitro studies and current clinical experience have indicated that resistant virus is slow to emerge and is transient.     

The action of Vibrio cholerae and Corynebacterium diphtheriae neuraminidases on the Sendai virus receptor of human erythrocytes.

Studies of the Sendai virus haemagglutinin receptor on the human erythrocyte surface have confirmed that it involves 2 leads to 3 linked sialic acid. Because the primary specificity of Vibrio cholerae neuraminidase is for this linkage, it is able to compete with the virus for the receptor, to which it adsorbs strongly at low temperatures. Corynebacterium diphtheriae neuraminidase, whose principal specificity is for a sialic acid linkage other than 2 leads to 3, does not easily remove Sendai virus receptors, nor does it adsorb to the erythrocyte surface. A new definition of the term "receptor-destroying enzyme" is given which takes both enzyme and virus specificity into account, and a modified assay method is suggested in order to overcome the problems due to enzyme adsorption.     

通用流感疫苗研究进展

流感是一种对人类危害极大的传染病,接种疫苗被认为是预防流感的最有效手段。目前所用的流感疫苗主要是根据现行流行株的减毒或灭活病毒疫苗及基于流感血凝素和神经氨酸酶设计的重组蛋白质疫苗。但流感病毒变异大,易逃逸机体免疫监视,有效的疫苗须不断分离新流行株和不断更新疫苗免疫原。为解决这一问题,很多科学家一直在研究基于病毒高度保守性蛋白质、能够预防所有流感病毒毒株、可诱导持久保护性免疫的通用流感疫苗。我们对基于基质蛋白M2、核蛋白等的通用流感疫苗做一简要综述。     

Heterosubtype Neutralizing Responses to Influenza A (H5N1) Viruses Are Mediated by Antibodies to Virus Haemagglutinin

Background

It is increasingly clear that influenza A infection induces cross-subtype neutralizing antibodies that may potentially confer protection against zoonotic infections. It is unclear whether this is mediated by antibodies to the neuraminidase (NA) or haemagglutinin (HA). We use pseudoviral particles (H5pp) coated with H5 haemagglutinin but not N1 neuraminidase to address this question. In this study, we investigate whether cross-neutralizing antibodies in persons unexposed to H5N1 is reactive to the H5 haemagglutinin.

Methodology/Principal Findings

We measured H5-neutralization antibody titers pre- and post-vaccination using the H5N1 micro-neutralization test (MN) and H5pp tests in subjects given seasonal vaccines and in selected sera from European elderly volunteers in a H5N1 vaccine trial who had detectable pre-vaccination H5N1 MN antibody titers. We found detectable (titer ≥20) H5N1 neutralizing antibodies in a minority of pre-seasonal vaccine sera and evidence of a serological response to H5N1 in others after seasonal influenza vaccination. There was excellent correlation in the antibody titers between the H5N1 MN and H5pp tests. Similar correlations were found between MN and H5pp in the pre-vaccine sera from the cohort of H5N1 vaccine trial recipients.

Conclusions/Significance

Heterosubtype neutralizing antibody to H5N1 in healthy volunteers unexposed to H5N1 is mediated by cross-reaction to the H5 haemagglutinin.     

Soluble recombinant influenza vaccines.

Soluble, recombinant forms of influenza A virus haemagglutinin and neuraminidase have been produced in cells of lower eukaryotes, and shown in a mouse model to induce complete protective immunity against a lethal virus challenge. Soluble neuraminidase, produced in a baculovirus system, consisted of tetramers, dimers and monomers. Only the tetramers were enzymatically active. The immunogenicity decreased very considerably in the order tetra > di > mono. Therefore, we fused the head part of the neuraminidase gene to a tetramerizing leucine zipper sequence; the resulting product was enzymatically active, tetrameric neuraminidase. The protective immunity induced by this engineered neuraminidase, however, remained fairly strain-specific. A third influenza A virus protein, the M2 protein, has only 23 amino acids exposed on the outer membrane surface. This extracellular part, M2e, has been remarkably conserved in all human influenza A strains since 1933. By fusing the M2e sequence to hepatitis B virus core protein, we could obtain highly immunogenic particles that induced complete, strain-independent, long-lasting protection in mice against a lethal viral challenge. Native M2 is a tetrameric protein and this conformation of the M2e part can also be mimicked by fusing this sequence to a tetramerizing leucine zipper. The potential of the resulting protein as a vaccine candidate remains to be evaluated.     

Purification of antibodies to influenza A virus structural proteins by affinity chromatography, and their participation in haemagglutination-inhibition, neutralization and enzyme-linked immunosorbent assay.

Affinity chromatography was used to purify rabbit antibodies to common and strain-specific antigeneic determinants of haemagglutinin, to neuraminidase, and to a combination of the internal proteins of influenza A viruses. The purity of the antibodies was assessed by haemagglutination-inhibition, enzyme-linked immunosorbent assay (ELISA) and competition ELISA. The antibodies were examined for their participation in neutralization and haemagglutination-inhibition assays, and in ELISA. ELISA was found to be the most sensitive technique for detecting antibodies. Antibodies to the common and strain-specific determinants of haemagglutinin were shown to participate in neutralization assay and ELISA, but only those antibodies to the common determinants were detected by haemagglutination-inhibition.     

Attachment, ingestion and intracellular killing of Helicobacter pylori by human peripheral blood mononuclear leukocytes and mouse peritoneal inflammatory macrophages

Abstract The different steps of phagocytosis, attachment, ingestion and intracellular killing of cells of Helicobacter pylori strain 17874 (expressing sialic acid-specific haemagglutinin) and cells of H. pylori strain 17875 (expressing non-sialic acid-specific haemagglutinin) have been studied. More cells of sialopositive H. pylori strain 17874 have been found attached to human peripheral blood mononuclear leukocytes (PBM) and mouse peritoneal inflammatory macrophages (PIM) than cells of sialonegative H. pylori strain 17875. Binding of cells of H. pylori strain 17874 has been significantly inhibited by treatment of phagocytes with neuraminidase. Inhibition of adhesion of these bacteria preincubated with foetuin to normal phagocytic cells has also been found. Well adhering cells of H. pylori strain 17874 were more resistant to killing mechanisms of human PBM and mouse PIM than cells of strain 17875. Good, probably sialic acid-specific haemagglutinin dependent, adhesion of H. pylori bacteria to phagocytes can be considered as an important virulence factor which facilitates the pathogen to avoid the defence mechanisms.     

Selection of predicted siRNA as potential antiviral therapeutic agent against influenza virus

Influenza virus A (IVA) infection is responsible for recent death worldwide. Hence, there is a need to develop therapeutic agents against the virus. We describe the prediction of short interfering RNA (siRNA) as potential therapeutic molecules for the HA (Haemagglutinin) and NA (Neuraminidase) genes. We screened 90,522 siRNA candidates for HA and 13,576 for NA and selected 1006 and 1307 candidates for HA and NA, respectively based on the proportion of viral sequences that are targeted by the corresponding siRNA, with complete matches. Further short listing to select siRNA with no off-target hits, fulfilling all the guidelines mentioned in approach, provided us 13 siRNAs for haemagglutinin and 13 siRNAs for neuraminidase. The approach of finding siRNA using multiple sequence alignments of amino acid sequences has led to the identification of five conserved amino acid sequences, three in hemagglutinin i.e. RGLFGAIAGFIE, YNAELLV and AIAGFIE and two in neuraminidase i.e. RTQSEC and EECSYP which on reveres translation provided siRNA sequences as potential therapeutic candidates. The approaches used during this study have enabled us to identify potentially therapeutic siRNAs against divergent IVA strains.     

Comparative structural analysis of haemagglutinin proteins from type A influenza viruses: conserved and variable features

Background

Genome variation is very high in influenza A viruses. However, viral evolution and spreading is strongly influenced by immunogenic features and capacity to bind host cells, depending in turn on the two major capsidic proteins. Therefore, such viruses are classified based on haemagglutinin and neuraminidase types, e.g. H5N1. Current analyses of viral evolution are based on serological and primary sequence comparison; however, comparative structural analysis of capsidic proteins can provide functional insights on surface regions possibly crucial to antigenicity and cell binding.

Results

We performed extensive structural comparison of influenza virus haemagglutinins and of their domains and subregions to investigate type- and/or domain-specific variation. We found that structural closeness and primary sequence similarity are not always tightly related; moreover, type-specific features could be inferred when comparing surface properties of haemagglutinin subregions, monomers and trimers, in terms of electrostatics and hydropathy. Focusing on H5N1, we found that variation at the receptor binding domain surface intriguingly relates to branching of still circulating clades from those ones that are no longer circulating.

Conclusions

Evidence from this work suggests that integrating phylogenetic and serological analyses by extensive structural comparison can help in understanding the ‘functional evolution’ of viral surface determinants. In particular, variation in electrostatic and hydropathy patches can provide molecular evolution markers: intriguing surface charge redistribution characterizing the haemagglutinin receptor binding domains from circulating H5N1 clades 2 and 7 might have contributed to antigenic escape hence to their evolutionary success and spreading.

Electronic supplementary material

The online version of this article (doi:10.1186/s12859-014-0363-5) contains supplementary material, which is available to authorized users.     

An enzymatic assay reveals that proteins destined for the apical or basolateral domains of an epithelial cell line share the same late Golgi compartments.

The expression of viral envelope proteins on the plasma membrane domains of the epithelial cell line, MDCK, is polar. Influenza virus infection of these cells leads to expression of the viral haemagglutinin and neuraminidase glycoproteins on the apical domain of the plasma membrane while vesicular stomatitis virus (VSV) infection yields basolateral expression of the sialic acid-bearing G protein. We have exploited the ability of the influenza neuraminidase to desialate the G protein of VSV to test for contact between these proteins during their intracellular transport to separate plasma membrane domains. We were able to select for VSV-G protein expression in doubly-infected cells because VSV protein production was accelerated in cells pre-infected with influenza virus. During double infection the envelope proteins of both viruses displayed the same polar localization as during single infection but the VSG-G protein was undersialated due to the action of the influenza neuraminidase. Incubation of singly-infected cells at 20 degrees C blocked the transport of VSV-G protein to the cell surface and resulted in increased sialation of the protein over that seen at 37 degrees C. This suggests that G protein is held in contact with the sialyl transferase at this temperature. 20 degrees C incubations of doubly-infected cells also produced the undersialated G protein characteristic of interaction with the neuraminidase. We conclude that most of the newly synthesised basolaterally-directed G protein is in physical contact with the majority of the neuraminidase through the terminal steps of Golgi processing.     

Phylogenetic Analysis of the Non-structural (NS) Gene of Influenza A Viruses Isolated in Kazakhstan in 2002-2009

Although the important role of the non-structural (NS1 and NEP) gene of influenza A in virulence of the virus is well established, our knowledge about the extent of variation in the NS gene pool of influenza A viruses in their natural reservoirs in Kazakhstan is incomplete. 17 influenza A viruses of different subtypes were studied in this paper. Seven types of haemagglutinin and five different neuraminidase subtypes in eight combinations were found among the isolated viruses. A comparison of nucleotide sequ...     

Purification and characterization of a novel haemagglutinin from Chlorella pyrenoidosa

We previously identified a strong haemagglutination activity in the freshwater unicellular green alga, Chlorella pyrenoidosa. Here, we sought to purify and characterize the haemagglutinin associated with this activity. Ammonium sulfate precipitation, gel filtration on sephacryl S-200 and DEAE-Sepharose ion-exchange chromatography were used to purify the haemagglutinin, which was designated CPH (Chlorella pyrenoidosa haemagglutinin). The molecular weight of CPH was estimated as 58 kDa by SDS-PAGE and 60 kDa by gel filtration of the native protein, indicating that this haemagglutinin exists as a monomer. The haemagglutinin activity of CPH was inhibited by glycoproteins, especially yeast mannan, but not by monosaccharides or disaccharides, indicating that CPH is carbohydrate-specific. In addition to the composition of CPH shown to be rich in glycine and acidic amino acids, heamagglutinating activity of CPH was insensitive to variations in pH or the presence of divalent cations, and atomic force microscopy revealed that the protein is rod-shaped. These results indicate that the characteristics of CPH are consistent with its identification as a haemagglutinin, and suggest that CPH may be a viable candidate for applications in a variety of biomedical fields.     

表达新城疫病毒HN基因的重组火鸡疱疹病毒的构建

应用聚合酶链反应技术,用设计带有限制性酶切位点的引物,特异地扩增从起始密码子开始的１ ．８ ｋｂ 新城疫病毒（ Ｎ Ｄ Ｖ） 血凝素神经氨酸酶（ Ｈ Ｎ） 基因开放式阅读框,然后插入ｐ Ｔ Ｋ２ Ｂ 的 Ｎｈｅ Ⅰ位点,构建了含 Ｎ Ｄ Ｖ Ｈ Ｎ 基因的插入载体ｐ Ｔ Ｋ Ｈ Ｎ１ 和ｐ Ｔ Ｋ Ｈ Ｎ２ ,再与感染火鸡疱疹病毒（ Ｈ Ｖ Ｔ） 细胞的总 Ｄ Ｎ Ａ 共转染鸡胚成纤维细胞（ Ｃ Ｅ Ｆ） ,经有限稀释法和 Ｄｏｔｂｌｏｔ 筛选,得到含有 Ｈ Ｎ 基因的重组体ｒ Ｈ Ｖ Ｔ１ 和ｒ Ｈ Ｖ Ｔ２ 。经组织培养传代和 Ｗｅｓｔｅｒｎ ｂｌｏｔ 分析,表明重组体在感染细胞中表达了 Ｈ Ｎ 蛋白。重组体在 Ｃ Ｅ Ｆ 上的生长特性与亲本病毒相同,且在连续传代过程中保持稳定。为国内新城疫基因工程疫苗研制奠定了基础。