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.     

Immunological studies with the HA1 and HA2 polypeptides of influenza A virus haemagglutinin.

HA1 and HA2 polypeptides of influenza A virus haemagglutinin (HA) were separated in purified form using electrophoresis in SDS containing polyacrylamide gels (PAGE) or chloroform-methanol extraction. The populations of HA1 polypeptides were immunogenic but considerably less so than the intact HA molecule and induced antibody which cross-reacted with influenza A and B viruses. After absorption with heterologous influenza B virus, the cross-reacting antibodies were removed and the HA1 antisera then possessed antibodies which reacted only with the cross-reactive (CR) determinants of the HA of the homologous influenza A virus and viruses of the same subtype. Neither strain-specific (SS) nor virus-neutralizing antibodies were detected in these anti-HA1 sera. HA2 polypeptides were less immunogenic and anti-HA2 antisera after absorption with influenza B virus failed to react with influenza A virus in immuno double diffusion tests and only reacted with partially denatured HA in the more sensitive single radial diffusion tests.     

Usefulness of ELISA and radial immunodiffusion tests for evaluation of the degree of purification of influenza diagnostic and vaccine preparations

It is necessary to use new diagnostic tests for careful and rapid evaluation of a degree of purification and immunogenicity of vaccine anti-influenza preparations. In this study in order to obtain this purpose a radial immunodiffusion++ test and immunoenzymatic test (ELISA) were used Recommended by WHO radial immunodiffusion++ test enable to determine a level of haemagglutinin of particular types and subtypes of influenza virus in polyvalent preparations. However, this test is time consuming therefore for hemagglutinin level determination ELISA test was adapted. This test is hundred times more sensitive and can be applied with success for determination of hemagglutinin level of influenza virus A or B. For evaluation of a degree of purification of vaccine preparations ELISA was elaborated, in which as an index of purification of preparation a level of ovalbumin is determined. This test is specific and extremely sensitive, and it is possible to determine ovalbumin level with accuracy of 1ng in 1 ml of preparation.     

Influenza Vaccine Manufacturing: Effect of Inactivation,Splitting and Site of Manufacturing. Comparison of Influenza Vaccine Production Processes

The aim of this study was to evaluate the impact of different inactivation and splitting procedures on influenza vaccine product composition, stability and recovery to support transfer of process technology. Four split and two whole inactivated virus (WIV) influenza vaccine bulks were produced and compared with respect to release criteria, stability of the bulk and haemagglutinin recovery. One clarified harvest of influenza H3N2 A/Uruguay virus prepared on 25.000 fertilized eggs was divided equally over six downstream processes. The main unit operation for purification was sucrose gradient zonal ultracentrifugation. The inactivation of the virus was performed with either formaldehyde in phosphate buffer or with beta-propiolactone in citrate buffer. For splitting of the viral products in presence of Tween^®, either Triton^™ X-100 or di-ethyl-ether was used. Removal of ether was established by centrifugation and evaporation, whereas removal of Triton-X100 was performed by hydrophobic interaction chromatography. All products were sterile filtered and subjected to a 5 months real time stability study. In all processes, major product losses were measured after sterile filtration; with larger losses for split virus than for WIV. The beta-propiolactone inactivation on average resulted in higher recoveries compared to processes using formaldehyde inactivation. Especially ether split formaldehyde product showed low recovery and least stability over a period of five months.     

A genetic and monoclonal analysis of high-yielding reassortants of influenza A virus used for human vaccines

This paper describes two methods of analysis using monoclonal antibodies and RNA hybridization to characterize variation in the haemagglutinins of seven high-yielding influenza virus reassortants used for inactivated vaccine production. The results show that variants' were selected in producing these genetic reassortants. The haemagglutinins of two reassortants showed both antigenic and structural differences from their wild-type (wt) parents as detected by the two methods of analysis. These variants were more closely related to other subtype strains which had previously been differentiated from the wt parent by post-infection ferret sera and which also had amino acid sequence differences in antigenically significant sites on the HA 1 polypeptide chain of the haemagglutinin molecule. The haemagglutinins of four of the seven reassortants showed antigenic differences but no apparent structural differences from their wt parents. The haemagglutinin of only on reassortant was antigenically and structurally identical to its wt parent. The variants could not be reliably distinguished with hyperimmune rabbit serum or immune ferret serum to the wt parent virus. It is therefore important to use more discriminatory tests to identify influenza strains correctly. It is also essential for vaccine strains to be as completely characterized as possible. It is considered desirable that both methods of analysis be used to characterize influenza virus reassortant strains.     

Expression of the influenza virus haemagglutinin in insect cells by a baculovirus vector.

The insect baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV) has played a major role in studies on the molecular biology of insect DNA viruses. Recently, this system has been effectively adapted as a highly efficient vector in insect cells for the expression of several mammalian genes. A cDNA sequence of the influenza (fowl plague) virus haemagglutinin gene has been inserted into the BamHI site of the pAc373 polyhedrin vector. Spodoptera frugiperda cells were co-transfected with this construct, pAc-HA651, and authentic AcNPV DNA. Recombinant virus was selected by adsorption of transfected cells to erythrocytes followed by serial plaque passages on S. frugiperda cells. We have determined the site of insertion of the haemagglutinin gene into the AcNPV genome by restriction enzyme cleavage and Southern blot hybridization analyses using haemagglutinin cDNA as a probe. The influenza haemagglutinin gene is located in the polyhedrin gene of AcNPV DNA. Immunofluorescent labelling, immunoprecipitation and immunoblot analyses with specific antisera revealed that S. frugiperda cells produce immune reactive haemagglutinin after infection with the recombinant virus. The haemagglutinin is expressed at the cell surface and has haemolytic capacity that has been activated by post-translational proteolytic cleavage. When chickens were immunized with S. frugiperda cells expressing haemagglutinin, they developed haemagglutinin-inhibiting and neutralizing antibodies and were protected from infection with fowl plague virus. These observations demonstrate that the haemagglutinin is processed in insect cells in a similar fashion as in fowl plaque virus-infected vertebrate cells and that it has full biological activity.     

Temperature and pH dependence of the haemolytic activity of influenza virus and of the rotational mobility of the spike glycoproteins

Influenza virus (strain X-47) was labeled with the triplet probe, eosin 5-isothiocyanate. Most of the label was found to be associated with haemagglutinin, the major glycoprotein of the viral envelope. Rotational diffusion of the glycoprotein was investigated by measuring flash-induced transient dichroism of the eosin probe. The anisotropy decay curves showed that mobility of haemagglutinin measured at pH 7.3 increased considerably with temperature with the greatest change occurring over the range 20-30 degrees C. However, at pH 5.2 no mobility was detectable over the time range of the experiment. The activity of the virus was determined by assaying haemolysis of human erythrocytes. The haemolytic activity showed an optimum at pH 5.2 and increased markedly with temperature, being negligible below 20 degrees C. In addition, inactivation of the virus by incubation at pH 5.2 was also strongly temperature dependent. A 15 min incubation at pH 5.2 inactivated the virus above 30 degrees C but had no effect below 20 degrees C. On the basis of these results, it is proposed that mobility of haemagglutinin is significant for its functional properties. When the pH is reduced from 7.3 to 5.2, the mobility observed at higher temperatures is required for the molecular rearrangements which accompany the fusion event. In the absence of an apposing membrane, these rearrangements result in irreversible aggregation of haemagglutinin in the viral membrane, and hence loss of mobility and activity.     

流行性感冒病毒血凝素亚单位疫苗在小鼠体内免疫效果观察

流感病毒表面抗原——血凝素(HA)亚单位,在人工合成的胞壁酰二肽(MDP)佐剂配合下,注射小白鼠所产生的免疫效果与常用的Al(OH)_3及福氏佐剂相似。含MDP佐剂的流感病毒HA亚单位疫苗腹腔注射小白鼠,能产生与福尔马林灭活的流感疫苗相似的免疫反应,而皮下注射,前者的免疫效果比后者明显为佳。     

Evolution of the Hong Kong influenza A sub-type. Structural relationships between the haemagglutinin from A/duck/Ukraine/1/63 (Hav 7) and the Hong Kong (H3) haemagglutinins.

The relationship between the haemagglutinin from the influenza virus A/duck/Ukraine/1/63 (Hav 7) and the human Hong Kong variants (H3) has been investigated. Amino-acid-sequence analysis shows that the Hav 7 haemagglutinin closely resembles the 1968 human H3 haemagglutinin in structure. However, the number of amino-acid-sequence differences (23) suggest that the Hong Kong haemagglutinin gene did not come directly from A/duck/Ukraine/1/63 but from a virus derived from it by antigenic drift during the period 1963-1968.     

Cell surface influenza haemagglutinin can mediate infection by other animal viruses.

We have used filter-grown Madin-Darby canine kidney (MDCK) cells to explore the mechanism by which influenza virus facilitates secondary virus infection. Vesicular stomatitis virus (VSV) and Semliki Forest virus (SFV) infect only through the basolateral surface of these polarized epithelial cells and not through the apical surface. Prior infection with influenza virus rendered the cell susceptible to infection by VSV or SFV through either surface. The presence of both a permissive and a restrictive surface for virus entry in the same cell allowed us to determine how the influenza infection enhanced the subsequent infection of a second virus. Biochemical and morphological evidence showed that influenza haemagglutinin on the apical surface serves as a receptor for the superinfecting virus by binding to its sialic acid-bearing envelope proteins. Influenza virus also facilitates secondary virus infection in non-epithelial cells; baby hamster kidney cells (BHK-21), which are normally resistant to infection by the coronavirus (mouse hepatitis virus MHV-A59), could be infected via the haemagglutinin-sialic acid interaction. Facilitation of secondary virus infection requires only the sialic acid-binding properties of the haemagglutinin since the uncleaved haemagglutinin could also mediate virus entry.     

Whole-genome sequences of influenza A(H3N2) viruses isolated from Brazilian patients with mild illness during the 2014 season

The influenza A(H3N2) virus has circulated worldwide for almost five decades and is the dominant subtype in most seasonal influenza epidemics, as occurred in the 2014 season in South America. In this study we evaluate five whole genome sequences of influenza A(H3N2) viruses detected in patients with mild illness collected from January-March 2014. To sequence the genomes, a new generation sequencing (NGS) protocol was performed using the Ion Torrent PGM platform. In addition to analysing the common genes, haemagglutinin, neuraminidase and matrix, our work also comprised internal genes. This was the first report of a whole genome analysis with Brazilian influenza A(H3N2) samples. Considerable amino acid variability was encountered in all gene segments, demonstrating the importance of studying the internal genes. NGS of whole genomes in this study will facilitate deeper virus characterisation, contributing to the improvement of influenza strain surveillance in Brazil.     

Application of deglycosylation to SDS PAGE analysis improves calibration of influenza antigen standards

Each year the production of seasonal influenza vaccines requires antigen standards to be available for the potency assessment of vaccine batches. These are calibrated and assigned a value for haemagglutinin (HA) content. The calibration of an antigen standard is carried out in a collaborative study amongst a small number of national regulatory laboratories which are designated by WHO as Essential Regulatory Laboratories (ERLs) for the purposes of influenza vaccine standardisation. The calibration involves two steps; first the determination of HA protein in a primary liquid standard by measurement of total protein in a purified influenza virus preparation followed by determination of the proportion of HA as determined by PAGE analysis of the sample; and second, the calibration of the freeze-dried reference antigen against the primary standard by single radial immunodiffusion (SRD) assay. Here we describe a collaborative study to assess the effect of adding a deglycosylation step prior to the SDS-PAGE analysis for the assessment of relative HA content. We found that while the final agreed HA value of the samples tested was not significantly different with or without deglycosylation, the deglycosylation step greatly improved between-laboratory agreement.     

The predicted antigenicity of the haemagglutinin of the 1918 Spanish influenza pandemic suggests an avian origin.

In 1982 we characterized the antigenic sites of the haemagglutinin of influenza A/PR/8/34, which is an influenza strain of the H1 subtype that was isolated from humans in 1934, by studying mutants which escaped neutralization by antibody. Four antigenic sites, namely Cb, Sa, Sb and Ca, were found to be located near the tip of the trimeric haemagglutinin spike. Based on the sequence of the haemagglutinin of the 1918 Spanish influenza, we can now specify the extent of divergence of antigenic sites of the haemagglutinin during the antigenic drift of the virus between 1918 and 1934. This divergence was much more extensive (40%) than the divergence (20%) in predicted antigenic sites between the 1918 Spanish influenza and an avian H1 subtype consensus sequence. These results support the hypothesis that the human 1918 pandemic originated from an avian virus of the H1 subtype that crossed the species barrier from birds to humans and adapted to humans, presumably by mutation and/or reassortment, shortly before 1918.     

A single radial hemolysis technique for the measurement of influenza virus antibody in swine serum

A single radial hemolysis technique was applied to the measurement of swine influenza virus antibody in swine serum. It was recognized to be useful for the detection of this antibody, although heating at 56 degrees C for 30 minutes was necessary to remove non--specific hemolysis from the test sample. The single radial hemolysis titer was correlative with the hemagglutination--inhibition titer in serum from pigs infected experimentally or naturally. This technique is simple, rapid and unaffected with non--specific inhibitors in swine serum. It does not require concentrated purified virus either. Therefore, it is applicable to the sero--epizootiologic surveillance of swine influenza on a large scale.     

Identifying Selection in the Within-Host Evolution of Influenza Using Viral Sequence Data

The within-host evolution of influenza is a vital component of its epidemiology. A question of particular interest is the role that selection plays in shaping the viral population over the course of a single infection. We here describe a method to measure selection acting upon the influenza virus within an individual host, based upon time-resolved genome sequence data from an infection. Analysing sequence data from a transmission study conducted in pigs, describing part of the haemagglutinin gene (HA1) of an influenza virus, we find signatures of non-neutrality in six of a total of sixteen infections. We find evidence for both positive and negative selection acting upon specific alleles, while in three cases, the data suggest the presence of time-dependent selection. In one infection we observe what is potentially a specific immune response against the virus; a non-synonymous mutation in an epitope region of the virus is found to be under initially positive, then strongly negative selection. Crucially, given the lack of homologous recombination in influenza, our method accounts for linkage disequilibrium between nucleotides at different positions in the haemagglutinin gene, allowing for the analysis of populations in which multiple mutations are present at any given time. Our approach offers a new insight into the dynamics of influenza infection, providing a detailed characterisation of the forces that underlie viral evolution.     

禽流感特异性转移因子的制备及其免疫作用

目的制备禽流感病毒特异性转移因子并探讨其对禽流感灭活疫苗的免疫增效作用。方法用禽流感病毒H5N1血清亚型灭活疫苗免疫鸡,用国标血凝抑制方法检测病毒特异性血凝抑制抗体效价。当抗体效价达到高峰时,翅静脉采取外周血,分离淋巴细胞并制备细胞单层、传代后获得禽流感病毒H5N1血清亚型特异性转移因子。用所获得的特异性转移因子进行疫苗免疫增效试验。结果采用本法可获得禽流感病毒特异性转移因子。免疫增效试验表明,在进行禽流感病毒灭活疫苗免疫的同时使用禽流感病毒特异性转移因子,可在一定幅度内提高禽流感病毒抗体水平并能延长抗体维持时间。不同给药途径比较试验表明,口服途径给药的疫苗增效作用优于注射途径给药。结论通过淋巴细胞体外培养可以制备禽流感病毒特异性转移因子。禽流感病毒H5N1血清亚型特异性转移因子对禽流感病毒灭活疫苗具有明显的增效作用,且口服途径给药的疫苗免疫增效作用优于注射途径给药。     

Intranasal immunization with inactivated influenza virus enhances immune responses to coadministered simian-human immunodeficiency virus-like particle antigens

Intranasal immunization with inactivated influenza virus vaccine can provide protective immunity, whereas many other antigens are less effective when used for mucosal immunization. To determine whether influenza virus could enhance immune responses to an antigen coadministered to a mucosal surface, we studied the intranasal immunization of mice with a mixture of simian-human immunodeficiency virus (SHIV) virus-like particles (VLPs) and inactivated influenza virus. Compared to mice immunized with SHIV VLPs alone, mice coimmunized with SHIV VLPs and inactivated influenza virus showed significant increases in serum immunoglobulin G (IgG) and mucosal IgA antibodies specific to the human immunodeficiency virus envelope protein, neutralizing activities, numbers of gamma interferon- and interleukin 4-secreting lymphocytes, and cytotoxic-T-lymphocyte activities. The levels of enhancement of immune response by coimmunization with inactivated influenza virus were equivalent to those induced by inclusion of immunostimulatory CpG oligodeoxynucleotides (CpG DNA). We also observed that SHIV VLPs bind to influenza virus virions, forming mixed aggregates. These results indicate that inactivated influenza virus can play a role as a mucosal adjuvant to coadministered antigens.     

Cationic lipid/DNA complex-adjuvanted influenza A virus vaccination induces robust cross-protective immunity

Influenza A virus is a negative-strand segmented RNA virus in which antigenically distinct viral subtypes are defined by the hemagglutinin (HA) and neuraminidase (NA) major viral surface proteins. An ideal inactivated vaccine for influenza A virus would induce not only highly robust strain-specific humoral and T-cell immune responses but also cross-protective immunity in which an immune response to antigens from a particular viral subtype (e.g., H3N2) would protect against other viral subtypes (e.g., H1N1). Cross-protective immunity would help limit outbreaks from newly emerging antigenically novel strains. Here, we show in mice that the addition of cationic lipid/noncoding DNA complexes (CLDC) as adjuvant to whole inactivated influenza A virus vaccine induces significantly more robust adaptive immune responses both in quantity and quality than aluminum hydroxide (alum), which is currently the most widely used adjuvant in clinical human vaccination. CLDC-adjuvanted vaccine induced higher total influenza virus-specific IgG, particularly for the IgG2a/c subclass. Higher levels of multicytokine-producing influenza virus-specific CD4 and CD8 T cells were induced by CLDC-adjuvanted vaccine than with alum-adjuvanted vaccine. Importantly, CLDC-adjuvanted vaccine provided significant cross-protection from either a sublethal or lethal influenza A viral challenge with a different subtype than that used for vaccination. This superior cross-protection afforded by the CLDC adjuvant required CD8 T-cell recognition of viral peptides presented by classical major histocompatibility complex class I proteins. Together, these results suggest that CLDC has particular promise for vaccine strategies in which T cells play an important role and may offer new opportunities for more effective control of human influenza epidemics and pandemics by inactivated influenza virus vaccine.     

Plasmid-only rescue of influenza A virus vaccine candidates.

The potential threat of another influenza virus pandemic stimulates discussion on how to prepare for such an event. The most reasonable prophylactic approach appears to be the use of effective vaccines. Since influenza and other negative-stranded RNA viruses are amenable to genetic manipulation using transfection by plasmids, it is possible to outline new reverse genetics-based approaches for vaccination against influenza viruses. We suggest three approaches. First, we use a plasmid-only rescue system that allows the rapid generation of high-yield recombinant vaccine strains. Second, we propose developing second-generation live influenza virus vaccines by constructing an attenuated master strain with deletions in the NS1 protein, which acts as an interferon antagonist. Third, we suggest the use of Newcastle disease virus recombinants expressing influenza virus haemagglutinin proteins of pandemic (epizootic) strains as novel vaccine vectors for use in animals and possibly humans.     

Crystallization and preliminary X-ray diffraction studies of a monoclonal antibody Fab fragment specific for an influenza virus haemagglutinin and of an escape mutant of that haemagglutinin

Preliminary crystallographic data are given for two molecules involved in the interaction between the humoral immune response and the influenza virus. These molecules are the Fab fragment of an antibody specific for the haemagglutinin of influenza virus strain X31 (Hong Kong 1/68 (H3N2)) and a mutant of X31 haemagglutinin that escapes recognition by that antibody. Crystals of the haemagglutinin are isomorphous to those of X31, whose structure is known; they diffract to 3.4 A resolution. Crystals of the Fab fragment are trigonal with space group P3(1)21 (or P3(2)21) and diffract to 2.6 A resolution. The unit cell dimensions are a = b = 98.9 A, c = 89.2 A. A native data set has been collected for both proteins.