Production and purification of chimeric HBc virus-like particles carrying influenza virus LAH domain as vaccine candidates.

BACKGROUND: The lack of a universal influenza vaccine is a global health problem. Interest is now focused on structurally conserved protein domains capable of eliciting protection against a broad range of influenza virus strains. The long alpha helix (LAH) is an attractive vaccine component since it is one of the most conserved influenza hemagglutinin (HA) stalk regions. For an improved immune response, the LAH domain from H3N2 strain has been incorporated into virus-like particles (VLPs) derived from hepatitis B virus core protein (HBc) using recently developed tandem core technology. RESULTS: Fermentation conditions for recombinant HBc-LAH were established in yeast Pichia pastoris and a rapid and efficient purification method for chimeric VLPs was developed to match the requirements for industrial scale-up. Purified VLPs induced strong antibody responses against both group 1 and group 2 HA proteins in mice. CONCLUSION: Our results indicate that the tandem core technology is a useful tool for incorporation of highly hydrophobic LAH domain into HBc VLPs. Chimeric VLPs can be successfully produced in bioreactor using yeast expression system. Immunologic data indicate that HBc VLPs carrying the LAH antigen represent a promising universal influenza vaccine component.

pH-triggered, activated-state conformations of the influenza hemagglutinin fusion peptide revealed by NMR.

The highly conserved first 23 residues of the influenza hemagglutinin HA2 subunit constitute the fusion domain, which plays a pivotal role in fusing viral and host-cell membranes. At neutral pH, this peptide adopts a tight helical hairpin wedge structure, stabilized by aliphatic hydrogen bonding and charge-dipole interactions. We demonstrate that at low pH, where the fusion process is triggered, the native peptide transiently visits activated states that are very similar to those sampled by a G8A mutant. This mutant retains a small fraction of helical hairpin conformation, in rapid equilibrium with at least two open structures. The exchange rate between the closed and open conformations of the wild-type fusion peptide is ~40 kHz, with a total open-state population of ~20%. Transitions to these activated states are likely to play a crucial role in formation of the fusion pore, an essential structure required in the final stage of membrane fusion.

Influenza viruses received and tested by the Melbourne WHO Collaborating Centre for Reference and Research on Influenza annual report, 2014.

The WHO Collaborating Centre for Reference and Research on Influenza in Melbourne is part of the World Health Organization's (WHO) Global Influenza Surveillance and Response System. In 2014 the Centre received a total of 5,374 influenza samples from laboratories primarily in the Asia-Pacific region. Viruses were characterised by their antigenic, genetic and antiviral drug resistance properties. Of the viruses successfully analysed 52% were A(H1N1)pdm09 viruses. The majority of these were antigenically and genetically similar to the WHO recommended reference strain for the 2014 Southern Hemisphere influenza vaccine. Results for A(H3N2) and B/Yamagata viruses suggested that circulating viruses of this subtype and lineage, respectively, had undergone antigenic and/or genetic changes, consistent with the decision by WHO to change recommended strains for the 2015 Southern Hemisphere vaccine. A small number of A(H1N1)pdm09 and B/Victoria viruses had highly reduced inhibition to the neuraminidase inhibitors oseltamivir and zanamivir. The Centre also undertook primary isolation of vaccine candidate viruses directly into eggs. A total of 38 viruses were successfully isolated in eggs, of which 1 (B/Phuket/3073/2013) was included in the 2015 Southern Hemisphere influenza vaccine.

[The separation, identification and molecular biologic analysis of isolates of pandemic influenza virus A(H1N1)pdm09].

The article presents data concerning analysis of bioassays under suspicion of influenza A(H1N1)pdm09. The technique of back transcription polymerase chain reaction and sequencing was applied. The separation of native influenza virus A was implemented using passaging through developing chick embryos.

Structure of coronavirus hemagglutinin-esterase offers insight into corona and influenza virus evolution.

The hemagglutinin-esterases (HEs) are a family of viral envelope glycoproteins that mediate reversible attachment to O-acetylated sialic acids by acting both as lectins and as receptor-destroying enzymes (RDEs). Related HEs occur in influenza C, toro-, and coronaviruses, apparently as a result of relatively recent lateral gene transfer events. Here, we report the crystal structure of a coronavirus (CoV) HE in complex with its receptor. We show that CoV HE arose from an influenza C-like HE fusion protein (HEF). In the process, HE was transformed from a trimer into a dimer, whereas remnants of the fusion domain were adapted to establish novel monomer-monomer contacts. Whereas the structural design of the RDE-acetylesterase domain remained unaltered, the HE receptor-binding domain underwent remodeling to such extent that the ligand is now bound in opposite orientation. This is surprising, because the architecture of the HEF site was preserved in influenza A HA over a much larger evolutionary distance, a switch in receptor specificity and extensive antigenic variation notwithstanding. Apparently, HA and HEF are under more stringent selective constraints than HE, limiting their exploration of alternative binding-site topologies. We attribute the plasticity of the CoV HE receptor-binding site to evolutionary flexibility conferred by functional redundancy between HE and its companion spike protein S. Our findings offer unique insights into the structural and functional consequences of independent protein evolution after interviral gene exchange and open potential avenues to broad-spectrum antiviral drug design.

Opposite polarity of virus budding and of viral envelope glycoprotein distribution in epithelial cells derived from different tissues.

We compared the surface envelope glycoprotein distribution and the budding polarity of four RNA viruses in Fischer rat thyroid (FRT) cells and in CaCo-2 cells derived from a human colon carcinoma. Whereas both FRT and CaCo-2 cells sort similarly influenza hemagglutinin and vesicular stomatitis virus (VSV) G protein, respectively, to apical and basolateral membrane domains, they differ in their handling of two togaviruses, Sindbis and Semliki Forest virus (SFV). By conventional EM Sindbis virus and SFV were shown to bud apically in FRT cells and basolaterally in CaCo-2 cells. Consistent with this finding, the distribution of the p62/E2 envelope glycoprotein of SFV, assayed by immunoelectronmicroscopy and by domain-selective surface biotinylation was predominantly apical on FRT cells and basolateral on CaCo-2 cells. We conclude that a given virus and its envelope glycoprotein can be delivered to opposite membrane domains in epithelial cells derived from different tissues. The tissue specificity in the polarity of virus budding and viral envelope glycoprotein distribution indicate that the sorting machinery varies considerably between different epithelial cell types.

Molecular dissection of radixin: distinct and interdependent functions of the amino- and carboxy-terminal domains.

The ERM proteins--ezrin, radixin, and moesin--occur in particular cortical cytoskeletal structures. Several lines of evidence suggest that they interact with both cytoskeletal elements and plasma membrane components. Here we described the properties of full-length and truncated radixin polypeptides expressed in transfected cells. In stable transfectants, exogenous full-length radixin behaves much like endogenous ERM proteins, localizing to the same cortical structures. However, the presence of full-length radixin or its carboxy-terminal domain in cortical structures correlates with greatly diminished staining of endogenous moesin in those structures, suggesting that radixin and moesin compete for a limiting factor required for normal associations in the cell. The results also reveal distinct roles for the amino- and carboxy-terminal domains. At low levels relative to endogenous radixin, the carboxy-terminal polypeptide is associated with most of the correct cortical targets except cleavage furrows. In contrast, the amino-terminal polypeptide is diffusely localized throughout the cell. Low level expression of full-length radixin or either of the truncated polypeptides has no detectable effect on cell physiology. However, high level expression of the carboxy-terminal domain dramatically disrupts normal cytoskeletal structures and functions. At these high levels, the amino-terminal polypeptide does localize to cortical structures, but does not affect the cells. We conclude that the behavior of radixin in cells depends upon activities contributed by separate domains of the protein, but also requires modulating interactions between those domains.

Hydrophobic-interaction chromatography for purification of influenza A and B virus.

Options for hydrophobic-interaction chromatography (HIC) for purification of cell culture-derived influenza A and B virus have been assessed using a 96-well plate format using a semi-high-throughput approach. Follow-up experiments at preparative scale were used to characterize dynamic binding capacity, viral hemagglutinin protein (HA protein) recovery, and the influence of influenza virus (IV) strains on yield and contamination levels. Virus recoveries of up to 96% with a residual DNA level of about 1.3% were achieved. To achieve DNA contamination levels required for manufacturing of influenza vaccines for human use, a purification train comprising clarification, inactivation, concentration, column-based anion-exchange chromatography (AEC), and HIC was used in a final set-up. AEC using strong quaternary ammonium ligands was applied as an orthogonal method for DNA depletion by adsorption. Subsequently, HIC (with polypropylene glycol as functional group) was used to reversibly bind virus particles for capture and to remove residual contaminating DNA and proteins (flow-through). This two-step chromatographic process, which requires neither a buffer exchange step nor nuclease treatment had a total virus particle yield for IV A/PR/8/34 (H1N1) of 92%. The protein and the DNA contamination level could be reduced to 42% and at least 1.0%, respectively. With 17.2 µg total protein and 2.0 ng DNA per monovalent dose, this purity level complies with the limits of the European Pharmacopeia for cell culture-derived human vaccines. Overall, the presented downstream process represents a valuable alternative to existing virus purification schemes. Furthermore, it utilizes only off-the-shelf materials and is a simple as well as an economic process for production of cell culture-derived viruses and viral vectors.

Influenza PR8 HA-specific Fab fragments produced by phage display methods.

Anti-influenza hemagglutinin (HA) Fabs were isolated from a phage display library using purified HA of influenza virus A/Puerto Rico/8/34 (PR8; H1N1) as an antigen. Four Fab clones displaying a 25-50-fold higher binding signal to PR8 HA than the control were selected for further analysis and comparison with anti-PR8 monoclonal antibody (mAb). All four Fabs and mAb recognized the PR8 HA under non-reducing conditions but rarely bound to reduced PR8 HA. Inhibition of influenza virus infection on MDCK cells was observed with Fab1 and mAb in a dose-dependent manner while Fab3 and 4 exhibited only a partial inhibitory effect. Moreover, Fab1 clone and mAb exhibited cross-reactivity with the A/Peking/262/95 (A/Peking; H1N1) strain. The inhibitory effects of mAb on both influenza strains were more potent than Fab1, which is likely attributed to its higher affinity for the antigen. SPR analyses, in fact, revealed that Fab1 and mAb have K(D) of 1.5 x 10(-8) and 3.2 x 10(-9)M, respectively. These results strongly suggest that phage library-derived Fabs can be readily prepared and that such HA-specific Fabs with inhibitory action on influenza infection may be used to treat influenza patients.

Heterologous expression and purification of the infectious salmon anemia virus hemagglutinin esterase.

This study presents the heterologous production and purification of a soluble and functional form of the hemagglutinin esterase (HE) of the infectious salmon anemia virus (ISAV) isolate 4 (Glesvaer/2/90). The HE possesses receptor binding and receptor destroying enzyme (RDE) activity and is probably involved in the infection process. The recombinant HE protein (recHE 4) was expressed in insect cells (Sf9) using the baculovirus expression vector system. Both the transmembrane region and the cytoplasmic tail were deleted, and a C-terminal His(6)-tag was attached to facilitate identification and purification of the recHE 4 protein. As determined by Western analysis the recHE 4 was secreted at 20 degrees C and not at 28 degrees C. By testing three HE constructs differing in their promoter and secretion signal sequences it was clear that the HE's own secretion signal sequence is more important than the promoter with respect to the amount of secreted recHE 4 obtained under the conditions used. A one-step purification by nickel-affinity chromatography resulted in a highly purified recHE 4, identified by matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) analysis. Also, the recHE 4 is glycosylated and contains disulfide bridges within the molecule. Functional studies including the verification of the receptor destroying enzyme (RDE) activity as well as the binding to Atlantic salmon erythrocytes (hemagglutination) indicate that the recHE 4 has similar functions as its native counterpart. In conclusion, insect cells secrete a functional form of the ISAV 4 HE. This is suitable for further analyses on its function and immunogenicity.

Characterization of influenza virus neuraminidase with hemagglutinin activity and its comparison with that of viral neuraminidase.

The neuraminidase associated with the bifunctional protein, hemagglutinin-neuraminidase, of influenza virus has been characterized. The enzyme has a pH optimum of 4.5, does not require Ca2+ and is inactivated (98%) by incubation at 50 degrees C. The enzyme has a Km of 2.00 X 10(-3) M and 0.06 X 10(-3) M with the substrates 2-(3-methoxyphenyl)-N-acetylneuraminic acid and fetuin, respectively. The Ki is 400 X 10(-6) with the inhibitor 2-deoxy-2,3-dehydro-N-acetylneuraminic acid. The incorporation of labeled cysteine, valine and leucine in the hemagglutinin-neuraminidase protein is different from that of viral neuraminidase. A comparison of the properties of the neuraminidase associated with protein hemagglutinin-neuraminidase with that of viral neuraminidase or sialidase showed that the former is biochemically different and an antigenically distinct enzyme. The unique feature of the new enzyme is that it has the hemagglutinin activity as well. The two biological activities could not be separated from each other in all systems used. Apparently, protein hemagglutinin-neuraminidase is genetically transferable and it is detectable in a laboratory recombinant virus E-2971 (H3 Aichi X N7). These results suggest that protein hemagglutinin-neuraminidase is a unique surface protein of the influenza virus A/Aichi/2/68 (H3N2).

Refinement of the influenza virus hemagglutinin by simulated annealing.

We have applied the method of simulated annealing to the refinement of the 3 A resolution crystal structure of the influenza virus hemagglutinin glycoprotein, using the program X-PLOR. Two different methods were introduced into X-PLOR to treat the non-crystallographic symmetry present in this and in other crystal structures. In the first, only the unique protomer atoms are refined; by application of the non-crystallographic symmetry operators to the protomer atoms, the X-ray structure factor derivatives are effectively averaged, and a non-bonded energy term models the interactions of the protomer with its neighbors in the oligomer without explicit refinement of the other protomers in the crystallographic asymmetric unit. In the second method, the entire asymmetric unit is refined, but an effective energy term is added to the empirical energy that restrains symmetry-related atomic positions to their average values after least-squares superposition. Several other modifications and additions were made to previously published X-PLOR protocols, including weighting of the X-ray terms, maintenance of the temperature of the molecular dynamics simulation, treatment of charged groups, changes in the values of certain empirical energy parameters, and the use of N-linked carbohydrate empirical energy parameters. The hemagglutinin refinement proceeded in several stages. An initial round of simulated annealing of the monomer was followed by rigid-body refinement of the 3-fold non-crystallographic symmetry axis position and a second round of monomer refinement. A third round was performed on the trimer using non-crystallographic symmetry restraints in all regions except those in lattice contacts showing obvious derivations from 3-fold symmetry. The refinement was completed with several rounds of conventional positional and isotropic temperature factor refinement needed to correct bad model geometry introduced by high-temperature molecular dynamics in regions of weak electron density. This structure was then used as the basis for refinement of three crystallographically isomorphous hemagglutinin structures, including complexes with the influenza virus receptor, sialic acid. Model geometry comparable to well-refined high-resolution structures was obtained with relatively little manual intervention, demonstrating the ability of simulated annealing refinement to produce highly idealized structures at moderate resolution.

Selective extraction of haemagglutinin and matrix protein from Sendai virions by employing trifluoperazine as a detergent.

Incubation of trifluoperazine, a local anaesthetic, at concentrations higher than the cmc with Sendai virus particles produces the selective solubilization of the haemagglutinin neuraminidase (HN) and matrix (M) proteins. This phenomenon involves aggregation of the Sendai virions and therefore the separation of HN and M from the rest of the particle can be performed by bench centrifugation. The supernatant contains the HN and M proteins and HN, once inserted into liposomes, elicits its own biological activities. Therefore, the method seems suitable for purifying large amounts of HN.

Systemic production of foreign peptides on the particle surface of tobacco mosaic virus.

By using a new tobacco mosaic virus (TMV) vector [Hamamoto, H., et al. (1993) Bio/Technology 11, 930-932], we have constructed TMV particles which present three different kinds of epitopes, two of them from influenza virus hemagglutinin (HA), and one from human immunodeficiency virus type I (HIV-I) envelope protein, on the surface of the particles. Each of these TMV particles reacted with each anti-peptide antiserum. These results suggest that this TMV vector can be used as an antigen presentation system.

Large-scale preparation of biologically active measles virus haemagglutinin expressed by attenuated vaccinia virus vectors.

A procedure described here allows the efficient and rapid purification of histidine-tagged measles virus haemagglutinin that is synthesized under the control of powerful promoters (PSFJ1-10 and PSFJ2-16) of the highly attenuated vaccinia virus (VV) strain LC16mO. A single affinity chromatography step purifies recombinant haemagglutinin proteins from the lysates of cells infected with the recombinant VVs. The recovery and purity are both very high (a yield of 0.5-2.8 mg/10(8) cells and purity of >94-98%), indicating that this procedure is approximately 400 times more efficient than the conventional methods used to prepare haemagglutinin. The haemagglutinins are correctly transported to the cell surface and have haemadsorption activity. Moreover, the recombinant haemagglutinin proteins cooperate with the measles virus fusion protein to elicit cell fusion activity. In addition, the antibody titres against measles virus, as measured by enzyme-linked immunosorbent assay using the purified haemagglutinin as the capture antigen, correlated closely with neutralization test titres (R(2) = 0.84, p < 0.05), indicating the preservation of immunologically relevant antigenicity. Such recombinant haemagglutinin preparations will be useful in diagnostic tests that measure functional anti-measles immunity and investigate the biological functions and structure of the haemagglutinin.

Sequence analysis of the equine H7 influenza virus haemagglutinin gene.

The nucleotide sequences of ten haemagglutinin genes of representative H7N7 equine influenza viruses isolated between 1956 and 1977 have been determined by primer extension sequencing. Their nucleotide and deduced amino acid sequences demonstrate a high degree of homology. These equine viruses can be divided into two distinct subgroups, the prototype-like, and a group comprising the early American isolates and the remaining equine viruses. The equine H7 haemagglutinins form a quite distinct group compared to H7 haemagglutinins isolated from other species. Each of these equine H7 haemagglutinins possess a tetrabasic amino acid cleavage site separating the HA1 and HA2 domains but, in addition, all ten contain a nine amino acid insertion prior to the tetrabasic sequence. The haemagglutinin glycoproteins of all ten viruses are capable of cleavage activation in virus infected primary chicken embryo fibroblast cells.

Characterization of baculovirus-expressed hemagglutinin and fusion glycoproteins of the attenuated measles virus strain AIK-C.

With measles virus cDNA of the avirulent vaccine strain AIK-C, two cDNAs of H or F genes were amplified by the polymerase chain reaction. The amplified cDNAs were inserted respectively to the baculovirus transfer vector pAcYM1 derived from the nuclear polyhedrosis virus of Autographa californica (AcNPV). After co-transfection of the transfer vectors with AcNPV DNA to Spodoptera frugiperda cells, recombinant baculoviruses were screened by plaque assay, and the viruses containing H-cDNA or F-cDNA were named H-AIK or F-AIK, respectively. By Western blot analyses, the band around 80 kDa and some smaller bands were appeared in the H-AIK infected S. frugiperda cells, and the band around 40 kDa was detected in the F-AIK infected cells. Immunofluorescence studies on unfixed S. frugiperda cells infected with H- or F-AIK recombinants showed that both antigens were transported to the cell surface. When green monkey red blood cells were added to the recombinant infected cells, H-AIK infected cells showed haemadsorption, and cells infected with F-AIK lysed the red blood cells. The recombinant proteins elicited the neutralizing antibodies against measles virus.

Rubella virosomes: preparation and ultrastructure.

Rubella virosomes were prepared from performed liposomes and detergent solubilized viral hemagglutinin. The liposomes were made from lecithin/dicetyl phosphate (3.5 : 1) films resuspended in NTE buffer and sonicated. Viral hemagglutinin was prepared from purified virus after solubilization with Triton X-100 and centrifugation through a sucrose gradient containing beta-D-octylglucoside. Electron microscopy of the rate zonal purified virosomes, showed virus-like structures of 40 - 80 nm. The virosomes retained the biological activity of the hemagglutinin and had a buoyant density of 1.2 g/cm3.

Purification of infectious rubella virus by gel filtration on sepharose 2B compared to gradient centrifugation in sucrose, sodium metrizoate and metrizamide.

Rubella virus was purified by chromatography on Sepharose 2B after concentration by ultrafiltration on hollow fibers and hydroextraction with PEG 20,000. Yields of 40% infectivity and 70% hemagglutinating activity were routinely obtained. Chromatographic purification was compared to ultracentrifugation in sucrose, metrizamide and sodium metrizoate. Yields were lower in sucrose and metrizamide, while sodium metrizoate reduced the infectivity of the virus below detectable levels. These results demonstrate the advantage of Sepharose 2B for the purification of infectious rubella virus.

Characterisation of rubella virus hemagglutinin rosettes.

Purified rubella virus treated with Triton X-100 was subjected to centrifugation in a sucrose density gradient containing nonionic detergent beta-D-octylglucoside. The result of this treatment was the formation of hemagglutinating rosettes containing viral glycoproteins VP2 (50,000 mol. wt.) and VP3 (63,000 mol. wt.). The rosettes have a 26 S sedimentation coefficient and a density of 1.2 g/cm3 in sucrose. Electron microscopy revealed 15 nm rosettes with a hollow center. The molecular weight of the rosettes was extrapolated at 850,000.