Antigen-specific production of immune interferon by T Cells lines.

Continuous cultures of T cells reactive to the hapten 4-ethoxymethylene-2-phenyloxazolone were tested for interferon production after antigenic stimulation in vitro. Induction of interferon was antigen-specific and also restricted by the I region of the major histocompatibility complex. Kinetics of antigen induced interferon production were different from those reported for mitogen induced synthesis.

Drug design against a shifting target: a structural basis for resistance to inhibitors in a variant of influenza virus neuraminidase.

BACKGROUND: Inhibitors of the influenza virus neuraminidase have been shown to be effective antiviral agents in humans. Several studies have reported the selection of novel influenza strains when the virus is cultured with neuraminidase inhibitors in vitro. These resistant viruses have mutations either in the neuraminidase or in the viral haemagglutinin. Inhibitors in which the glycerol sidechain at position 6 of 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (Neu5Ac2en) has been replaced by carboxamide-linked hydrophobic substituents have recently been reported and shown to select neuraminidase variants. This study seeks to clarify the structural and functional consequences of replacing the glycerol sidechain of the inhibitor with other chemical constituents. RESULTS: The neuraminidase variant Arg292-->Lys is modified in one of three arginine residues that encircle the carboxylate group of the substrate. The structure of this variant in complex with the carboxamide inhibitor used for its selection, and with other Neu5Ac2en analogues, is reported here at high resolution. The structural consequences of the mutation correlate with altered inhibitory activity of the compounds compared with wild-type neuraminidase. CONCLUSIONS: The Arg292-->Lys variant of influenza neuraminidase affects the binding of substrate by modification of the interaction with the substrate carboxylate. This may be one of the structural correlates of the reduced enzyme activity of the variant. Inhibitors that have replacements for the glycerol at position 6 are further affected in the Arg292-->Lys variant because of structural changes in the binding site that apparently raise the energy barrier for the conformational change in the enzyme required to accommodate such inhibitors. These results provide evidence that a general strategy for drug design when the target has a high mutation frequency is to design the inhibitor to be as closely related as possible to the natural ligands of the target.

The structure of a complex between the NC10 antibody and influenza virus neuraminidase and comparison with the overlapping binding site of the NC41 antibody.

BACKGROUND: While it is well known that different antibodies can be produced against a particular antigen, and even against a particular site on an antigen, up until now there have been no structural studies of cross-reacting antibodies of this type. One antibody-antigen complex whose structure is known is that of the influenza virus antigen, neuraminidase, in complex with the NC41 antibody. Another anti-neuraminidase antibody, NC10, binds to an overlapping site on the antigen. The structure of the complex formed by this antibody with neuraminidase is described here and compared with the NC41-containing complex. RESULTS: The crystal structure of the NC10 Fab-neuraminidase complex has been refined to a nominal resolution of 2.5A. Approximately 80% of the binding site of the NC10 antibody on neuraminidase overlaps with that of the NC41 antibody. The epitope residues of neuraminidase are often engaged in quite different interactions with the two antibodies. Although the NC10 and NC41 antibodies have identical amino acid sequences within the first complementarity determining region of their heavy chains, this is not the basis of the cross-reaction. CONCLUSIONS: The capacity of two different proteins to bind to the same target structure on a third protein need not be based on the existence of identical or homologous amino acid sequences within those proteins. As we have demonstrated, amino acid residues on the common target structure may be in quite different chemical environments, and may also adopt different conformations within two protein-protein complexes.

The structure of the fusion glycoprotein of Newcastle disease virus suggests a novel paradigm for the molecular mechanism of membrane fusion.

BACKGROUND: Membrane fusion within the Paramyxoviridae family of viruses is mediated by a surface glycoprotein termed the "F", or fusion, protein. Membrane fusion is assumed to involve a series of structural transitions of F from a metastable (prefusion) state to a highly stable (postfusion) state. No detail is available at the atomic level regarding the metastable form of these proteins or regarding the transitions accompanying fusion. RESULTS: The three-dimensional structure of the fusion protein of Newcastle disease virus (NDV-F) has been determined. The trimeric NDV-F molecule is organized into head, neck, and stalk regions. The head is comprised of a highly twisted beta domain and an additional immunoglobulin-like beta domain. The neck is formed by the C-terminal extension of the heptad repeat region HR-A, capped by a four-helical bundle. The C terminus of HR-A is encased by a further helix HR-C and a 4-stranded beta sheet. The stalk is formed by the remaining visible portion of HR-A and by polypeptide immediately N-terminal to the C-terminal heptad repeat region HR-B. An axial channel extends through the head and neck and is fenestrated by three large radial channels located approximately at the head-neck interface. CONCLUSION: We propose that prior to fusion activation, the hydrophobic fusion peptides in NDV-F are sequestered within the radial channels within the head, with the central HR-A coiled coil being only partly formed. Fusion activation then involves, inter alia, the assembly of a complete HR-A coiled coil, with the fusion peptides and transmembrane anchors being brought into close proximity. The structure of NDV-F is fundamentally different than that of influenza virus hemagglutinin, in that the central coiled coil is in the opposite orientation with respect to the viral membrane.

The use of tetramethylbenzidine for solid phase immunoassays.

A rapid, sensitive, solid-phase immunoassay, using horseradish peroxidase and 3,3',5,5'-tetramethylbenzidine, was found to be more sensitive and the colour developed was more stable compared with HRPO using 4-chloro-1-naphthol or diaminobenzidine. Assay sensitivity was equal to or greater than that obtained with the alkaline phosphatase reaction. The essential step was to incubate filters in 1% dextran sulphate for 10 min in a pH 5 (10 mM citrate-EDTA) buffer before reacting with TMB (0.05 mg/ml in the same buffer).

Preparation of T cell growth factor free from interferon and factors stimulating hemopoietic cells and mast cells.

A simple two-step method involving ammonium sulfate precipitation followed by hydrophobic chromatography is described for the separation of T cell growth factor (TCGF) from a number of other factors contained in medium conditioned by concanavalin A-stimulated spleen cells. Thus, granulocyte-macrophage colony-stimulating factor, P cell-stimulating activity, pluripotential stem cell-supporting activity and interferon activity were not detected in TCGF partially purified by these steps. T cell-replacing factor co-purified with TCGF. Macrophage activity factor (MAF) co-purified with TCGF, but the ratio of MAF to TCGF activities was reduced more than 20-fold relative to that in crude conditioned medium. All of the factors were present in the 50-80% saturated ammonium sulfate fraction, however, levels of concanavalin A were reduced by 98% in this step. TCGF, separated in this way from these other regulatory factors will be useful in experiments analyzing the actions of TCGF on mixed populations of cells.

Liquid nitrogen storage of cultured T lymphocytes.

The conditions required for storing and recovering IL-2-dependent T cell clones from liquid nitrogen were investigated. For maximum cell recovery, cultured T lymphocytes were precooled at 4 degrees C for 15 min in medium containing 10% DMSO and 20% FCS before storage in liquid nitrogen. This method allowed adequate time for DMSO to penetrate the cells before freezing.

Affinity ranking of influenza neuraminidase mutants with monoclonal antibodies using an optical biosensor. Comparison with ELISA and slot blot assays.

A recently developed alternative to the more traditional techniques for studying antigen-antibody interactions has been examined. This method involves the use of an optical biosensor employing surface plasmon resonance detection. In this system one of the reactants is immobilized on the sensor surface and other reactants are passed over the sensor surface sequentially at a constant flow rate. Binding phenomena are detected in real time from changes in the angle at which surface plasmon resonance occurs. This is dependent, among other things, on changes in the refractive index (which is directly proportional to the mass) at or near to the sensor surface. Applications of this biosensor technique for comparing the binding of related neuraminidases, purified from escape mutants of influenza virus NWS/G70C/75 (N9), to two antibody Fab fragments, are described. These results were compared with those obtained from ELISA and slot blot assays on the same neuraminidases interacting with the same two monoclonal antibodies. The biosensor method was shown to be highly specific, permitting rapid screening of binding in such antigen-antibody systems.

Rapid treatment of whole cells and RNA viruses for analysis of RNA by slot blot hybridization.

To avoid extensive manipulation for the purification of RNA from cells, several methods were evaluated for the direct release of RNA from influenza virus infected cells and supernatants using slot blot hybridization and non-radioactive probes. Treatment with an equal volume of 10 M aqueous guanidine hydrochloride produced the best hybridization signal. Less, but significant amounts of RNA were also released using the following treatments: dilute alkali (final concentration of 0.16 M NaOH) or 100 degrees C/5 min or RNA sample buffer containing formamide/formaldehyde, then heating at 65 degrees C/10 min. Despite the presence of large amounts of cell debris, RNA from guanidine hydrochloride treated whole cell extracts bound quantitatively to the positively charged nylon membranes. The sensitivity of RNA detection when whole cell extracts treated with guanidine hydrochloride were probed with a digoxigenin labelled cDNA probe was similar to the detection of RNA in highly purified, protein free samples. Three positively charged membranes were tested (from Amersham, ICN and Boehringer Mannheim) using two alkaline phosphatase substrates, NBT-X phos, and a chemiluminescent substrate, 3-(2'-spiroadamantane)-4-methoxy-4-(3'-phosphoyloxy)-phenyl- 1-1,2-dioextane (AMPPD) and a peroxidase substrate, tetramethylbenzidine (TMB). The Boehringer Mannheim membrane had the highest sensitivity for the alkaline phosphatase substrates, but the peroxidase reaction with the TMB substrate was the most consistently sensitive, irrespective of which membrane was used. The ability to quantitatively detect RNA from whole cells without any purification will allow the rapid screening of large numbers of samples for specific RNA species in research or diagnostic laboratories.

Expression of influenza neuraminidase in baculovirus-infected cells.

Recombinant influenza neuraminidase (NA, subtype 2, A/NT/60/68) was produced by recombinant baculovirus-infected insect cells. The recombinant NA retained enzyme activity and was located on the cell surface. Enzyme activity was both cell-associated and in the cell free supernatant; maximal NA activity was found in the supernatant. Recombinant NA was recognised by polyclonal antisera and by three monoclonal antibodies specific for NA (subtype 2). Enzyme activity was also neutralised by polyclonal antisera. Recombinant NA thus retains most of the immunological and activity properties of authentic influenza NA. Immunoprecipitation of [35S]Methionine-labelled cells and supernatant and partial purification of NA indicated that a approximately 50-kDa form of NA was present in the supernatant, whilst the expected size (approximately 67-kDa) was cell-associated. Purified recombinant extracellular virus was also enzymatically active, and contained the 67-kDa NA which was located on the membrane capsule of the virus. This suggests that the virus had acquired the cell-associated form of recombinant NA during the budding process from the cell.

VP-14637 ViroPharma.

VP-14637 is the lead compound in a series of low molecular weight viral replication inhibitors which are under preclinical investigation by ViroPharma for the potential treatment of RSV infection [322651]. Phase I trials designed to evaluate the safety and pharmacokinetic prpfile of VR-14637 in healthy human volunteers have begun [385870]. VP-14637 is most active against pneumoviruses and the available data suggest that it is an inhibitor of RSV viral fusion activity [363716], [361097].

Use of oligonucleotide probes for selecting potential high-yielding influenza reassortants.

A new method for rapid screening of high-yielding reassortants of influenza virus, as candidates for vaccine production, is described. Oligonucleotide probes specific for all the parent genes of A/PR/8/34 (PR8), except the HA and the NA were designed based on database information available for different influenza strains. Digoxigenin labelled probes were tested by slot-blot hybridizations to purified RNA from a panel of A/PR/8/34 wild type and A/PR/8/34 reassortant viruses. The results show that the vast majority of reassortants selected for their high growth yield had acquired the non-structural (NS), matrix (M) and RNA polymerase 2 (PB2) genes from the PR8 parent. It is proposed that probes for these genes provide the potential for a simple and rapid procedure for selection of candidate high-yield reassortants for vaccine production.

A new method for the purification of the influenza A virus neuraminidase.

A rapid new method for the purification of neuraminidase (NA) heads from influenza A virus is described. Virus was pelleted directly from allantoic fluid and was digested with pronase. The cores were removed by centrifugation, redigested and the released NA heads were pooled and concentrated. The NA was separated from all contaminating proteins in a single step on a Superose 12 column. The purified material was suitable for both crystallography and for the production of monospecific antisera.

Biochemical Methods for the Characterization of Influenza Viruses with Reduced Sensitivity to 4-Guanidino-Neu5Ac2en.

Viruses that are less sensitive to the influenza neuraminidase (NA)-specific inhibitor 4-guanidino-Neu5Ac2en (zanamavir) (1) can be isolated after several passages in MDCK cells in the presence of the inhibitor. Although there are three reports of a mutation in the NA gene at the same conserved site, glu119 (2-4), most of the variants have mutations in the hemagglutinin (HA) gene (5). Many of these mutations appear to lower the affinity of the HA for the cellular receptor, so there is less requirement for significant NA activity for the newly synthesized progeny virus to elute. In this chapter we describe noncell culture-based methods for characterization of both HA and NA variants.

Virological Methods for the Generation and Characterization of Influenza Viruses with Reduced Sensitivity to 4-Guanidino-Neu5Ac2en.

The compound 4-guanidino-Neu5Ac2en (zanamivir) has been described as a selective inhibitor of the influenza virus neuraminidase (NA) (1). Viruses that are less sensitive to this inhibitor can be isolated after several passages in MDCK cells in the presence of the inhibitor. Variants isolated so far have had mutations predominantly in the hemagglutinin (HA) gene (2). Many of these mutations appear to lower the affinity of the HA for the cellular receptor, so that there is less requirement for significant NA activity for the newly synthesized progeny virus to elute. There are three reports of a mutation in the NA gene, all at the same conserved site, glu 119 (3-5). In this chapter, the authors describe methods for the isolation of the mutants, and for their characterization in cell culture based assays.

Isolation of very virulent Marek's disease viruses from vaccinated chickens in Australia.

Very virulent Marek's disease viruses (vvMDV), defined as isolates against which the herpesvirus of turkey (HVT) vaccine provide poor protection, have been isolated from poultry flocks in both the United States and Europe. Twenty-one samples from vaccinated Australian flocks, experiencing problems with excessive Marek's disease (MD), were tested for the presence of transmissible MD viruses (MDV). Of the 16 samples which contained a transmissible agent, 14 were pathogenic in chickens, based on the development of MD lesions or depression of the bursa/body weight ratio. Of the pathogenic isolates which have been successfully typed 10 were serotype 1, and one was serotype 2 MDV. Pathogenicity of isolates varied. Several isolates caused tumours in 20-30% of both vaccinated and unvaccinated chickens. Two isolates, MPF6 and MPF23, caused tumours in more than 50% of chickens. When MPF6 and MPF23 were tested in vaccine trials bivalent vaccine gave no better protection against development of MD lesions than a monovalent vaccine. Isolate MPF23 was so pathogenic that lesions were produced in all chickens, regardless of the vaccine protocol used. Therefore vvMDV have been isolated in Australia, and unlike the vaccines tested overseas, bivalent Australian vaccines do not appear to provide greater protection against these vvMDV.

Resistance to neuraminidase inhibitors conferred by an R292K mutation in a human influenza virus H7N9 isolate can be masked by a mixed R/K viral population.

UNLABELLED: We characterized the A/Shanghai/1/2013 virus isolated from the first confirmed human case of A/H7N9 disease in China. The A/Shanghai/1/2013 isolate contained a mixed population of R (65%; 15/23 clones) and K (35%; 8/23 clones) at neuraminidase (NA) residue 292, as determined by clonal sequencing. A/Shanghai/1/2013 with mixed R/K at residue 292 exhibited a phenotype that is sensitive to zanamivir and oseltamivir carboxylate by the enzyme-based NA inhibition assay. The plaque-purified A/Shanghai/1/2013 with dominant K292 (94%; 15/16 clones) showed sensitivity to zanamivir that had decreased by >30-fold and to oseltamivir carboxylate that had decreased by >100-fold compared to its plaque-purified wild-type counterpart possessing dominant R292 (93%, 14/15 clones). In Madin-Darby canine kidney (MDCK) cells, the plaque-purified A/Shanghai/1/2013-NAK292 virus exhibited no reduction in viral titer under conditions of increasing concentrations of oseltamivir carboxylate (range, 0 to 1,000 µM) whereas the replication of the plaque-purified A/Shanghai/1/2013-NAR292 and the A/Shanghai/2/2013 viruses was completely inhibited at 250 µM and 31.25 µM of oseltamivir carboxylate, respectively. Although the plaque-purified A/Shanghai/1/2013-NAK292 virus exhibited lower NA enzyme activity and a higher Km for 2'-(4-methylumbelliferryl)-α-d-N-acetylneuraminic acid than the wild-type A/Shanghai/1/2013-NAR292 virus, the A/Shanghai/1/2013-NAK292 virus formed large plaques and replicated efficiently in vitro. Our results confirmed that the NA R292K mutation confers resistance to oseltamivir, peramivir, and zanamivir in the novel human H7N9 viruses. Importantly, detection of the resistance phenotype may be masked in the clinical samples containing a mixed population of R/K at NA residue 292 in the enzyme-based NA inhibition assay. IMPORTANCE: The neuraminidase (NA) inhibitors oseltamivir and zanamivir are currently the front-line therapeutic options against the novel H7N9 influenza viruses, which possess an S31N mutation that confers resistance to the M2 ion channel blockers. It is therefore important to evaluate the sensitivity of the clinical isolates to NA inhibitors and to monitor for the emergence of resistant variants. We characterized the A/Shanghai/1/2013 (H7N9) isolate which contained a mixed population of R/K at NA residue 292. While the clinical isolate exhibited a phenotype of sensitivity to NA inhibitors using the enzyme-based NA inhibition assay, the plaque-purified A/Shanghai/1/2013 virus with dominant K292 was resistant to zanamivir, peramivir, and oseltamivir. Resistance to NA inhibitors conferred by the R292K mutation in a human influenza virus H7N9 isolate can be masked by a mixed R/K viral population, and this should be taken into consideration while monitoring antiviral resistance in patients with H7N9 infection.