Non-promoting diterpene esters can induce Epstein-Barr Virus early antigen expression in the Raji cell line

A range of diterpene ester ligands with selective biological activity (e.g., irritant but not tumour promoting) were tested for their ability to induce Epstein-Barr virus (EBV) early antigen expression in the lymphoblastoid Raji cell line. All substituted compounds were found to be capable of inducing some antigen expression at nM−μM levels, including desacetyl-α-sapinine, a compound largely devoid of biological activity. The non-promoting, fluorescent compound, sapintoxin A, was virtually equipotent with promoting compounds. It was concluded that, although the assay has relevance to the specific condition of chronic diterpene ester exposure occurring in conjunction with high EBV infection rates, there was relatively poor correlation with mouse skin tumour promoting potential.

Inhibition of coxsackie B virus infection by soluble forms of its receptors: Binding affinities, altered particle formation, and competition with cellular receptors

We previously reported that soluble decay-accelerating factor (DAF) and coxsackievirus-adenovirus receptor (CAR) blocked coxsackievirus 133 (CVB3) myocarditis in mice, but only soluble CAR blocked CVB3-mediated pancreatitis. Here, we report that the in vitro mechanisms of viral inhibition by these soluble receptors also differ. Soluble DAF inhibited virus infection through the formation of reversible complexes with CVB3, while binding of soluble CAR to CVB induced the formation of altered (A) particles with a resultant irreversible loss of infectivity. A-particle formation was characterized by loss of VP4 from the virions and required incubation of CVB3-CAR complexes at 37 degrees C. Dimeric soluble DAF (DAF-Fc) was found to be 125-fold-more effective at inhibiting CVB3 than monomeric DAF, which corresponded to a 100-fold increase in binding affinity as determined by surface plasmon resonance analysis. Soluble CAR and soluble dimeric CAR (CAR-Fc) bound to CVB3 with 5,000- and 10,000-fold-higher affinities than the equivalent forms of DAF. While DAF-Fc was 125-fold-more effective at inhibiting virus than monomeric DAF, complement regulation by DAF-Fc was decreased 4 fold. Therefore, while the virus binding was a cooperative event, complement regulation was hindered by the molecular orientation of DAF-Fc, indicating that the regions responsible for complement regulation and virus binding do not completely overlap. Relative contributions of CVB binding affinity, receptor binding footprint on the virus capsid, and induction of capsid conformation alterations for the ability of cellular DAF and CAR to act as receptors are discussed.

Rational engineering of recombinant picornavirus capsids to produce safe, protective vaccine antigen

Foot-and-mouth disease remains a major plague of livestock and outbreaks are often economically catastrophic. Current inactivated virus vaccines require expensive high containment facilities for their production and maintenance of a cold-chain for their activity. We have addressed both of these major drawbacks. Firstly we have developed methods to efficiently express recombinant empty capsids. Expression constructs aimed at lowering the levels and activity of the viral protease required for the cleavage of the capsid protein precursor were used; this enabled the synthesis of empty A-serotype capsids in eukaryotic cells at levels potentially attractive to industry using both vaccinia virus and baculovirus driven expression. Secondly we have enhanced capsid stability by incorporating a rationally designed mutation, and shown by X-ray crystallography that stabilised and wild-type empty capsids have essentially the same structure as intact virus. Cattle vaccinated with recombinant capsids showed sustained virus neutralisation titres and protection from challenge 34 weeks after immunization. This approach to vaccine antigen production has several potential advantages over current technologies by reducing production costs, eliminating the risk of infectivity and enhancing the temperature stability of the product. Similar strategies that will optimize host cell viability during expression of a foreign toxic gene and/or improve capsid stability could allow the production of safe vaccines for other pathogenic picornaviruses of humans and animals.

Contribution of redox status to hepatitis C virus E2 envelope protein function and antigenicity

Disulfide bonding contributes to the function and antigenicity of many viral envelope glycoproteins. We assessed here its significance for the hepatitis C virus E2 envelope protein and a counterpart deleted for hypervariable region-1 (HVR1). All 18 cysteine residues of the antigens were involved in disulfides. Chemical reduction of up to half of these disulfides was compatible with anti-E2 monoclonal antibody reaction, CD81 receptor binding, and viral entry, whereas complete reduction abrogated these properties. The addition of 5,5'-dithiobis-2-nitrobenzoic acid had no effect on viral entry. Thus, E2 function is only weakly dependent on its redox status, and cell entry does not require redox catalysts, in contrast to a number of enveloped viruses. Because E2 is a major neutralizing antibody target, we examined the effect of disulfide bonding on E2 antigenicity. We show that reduction of three disulfides, as well as deletion of HVR1, improved antibody binding for half of the patient sera tested, whereas it had no effect on the remainder. Small scale immunization of mice with reduced E2 antigens greatly improved serum reactivity with reduced forms of E2 when compared with immunization using native E2, whereas deletion of HVR1 only marginally affected the ability of the serum to bind the redox intermediates. Immunization with reduced E2 also showed an improved neutralizing antibody response, suggesting that potential epitopes are masked on the disulfide-bonded antigen and that mild reduction may increase the breadth of the antibody response. Although E2 function is surprisingly independent of its redox status, its disulfide bonds mask antigenic domains. E2 redox manipulation may contribute to improved vaccine design.

The molecular basis of HIV capsid assembly - five years of progress

The assembly of HIV is relatively poorly investigated when compared with the process of virus entry. Yet a detailed understanding of the mechanism of assembly is fundamental to our knowledge of the complete life cycle of this virus and also has the potential to inform the development of new antiviral strategies. The repeated multiple interaction of the basic structural unit, Gag, might first appear to be little more than concentration dependent self-assembly but the precise mechanisms emerging for HIV are far from simple. Gag interacts not only with itself but also with host cell lipids and proteins in an ordered and stepwise manner. It binds both the genomic RNA and the virus envelope protein and must do this at an appropriate time and place within the infected cell. The assembled virus particle must successfully release from the cell surface and, whilst being robust enough for transmission between hosts, must nonetheless be primed for rapid disassembly when infection occurs. Our current understanding of these processes and the domains of Gag involved at each stage is the subject of this review. Copyright (C) 2004 John Wiley Sons, Ltd.

Restrictions to the adaptation of influenza A virus H5 hemagglutinin to the human host

The binding specificities of a panel of avian influenza virus subtype H5 hemagglutinin (RA) proteins bearing mutations at key residues in the receptor binding site were investigated. The results demonstrate that two simultaneous mutations in the receptor binding site resulted in H5 RA binding in a pattern similar to that shown by human viruses. Coexpression of the ion channel protein, M2, from most avian and human strains tested protected H5 RA conformation during trafficking, indicating that no genetic barrier to the reassortment of the H5 surface antigen gene with internal genes of human viruses existed at this level.

A block in virus-like particle maturation following assembly of murine leukaemia virus in insect cells

Expression of the murine leukaemia virus (MLV) major Gag antigen p65(Gag) using the baculovirus expression system leads to efficient assembly and release of virus-like particles (VLP) representative of immature MLV. Expression of P180(Gag-Pol), facilitated normally in mammalian cells by readthrough of the p65(Gag) termination codon, also occurs efficiently in insect cells to provide a source of the MLV protease and a pattern of p65(Gag) processing similar to that observed in mammalian cells. VLP release from P180(Gag-Pol) expressing cells however remains essentially immature with disproportionate levels of the uncleaved p65(Gag) precursor when compared to the intracellular Gag profile. Changing the p65(Gag) termination codon altered the level of p65(Gag) and p180(Gag-Pol) within expressing cells but did not alter the pattern of released VLP, which remained immature. Coexpression of p65(Gag) with a fixed readthrough p180(Gag-Pol) also led to only immature VLP release despite high intracellular protease levels. Our data suggest a mechanism that preferentially selects uncleaved p65(Gag) for the assembly of MLV in this heterologous expression system and implies that, in addition to their relative levels, active sorting of the correct p65(Gag) and p180(Gag-Pol) ratios may occur in producer cells. (C) 2003 Elsevier Inc. All rights reserved.

Evaluation of a nucleoprotein-based enzyme-linked immunosorbent assay for the detection of antibodies against infectious bronchitis virus

As an immunogen of the coronavirus, the nucleoprotein (N) is a potential antigen for the serological monitoring of infectious bronchitis virus (IBV). In this report, recombinant N protein from the Beaudette strain of IBV was produced and purified from Escherichia coli as well as Sf9 ( insect) cells, and used for the coating of enzyme-linked immunosorbent assay ( ELISA) plates. The N protein produced in Sf9 cells was phosphorylated whereas N protein from E. coli was not. Our data indicated that N protein purified from E. coli was more sensitive to anti-IBV serum than the protein from Sf9 cells. The recombinant N protein did not react with the antisera to other avian pathogens, implying that it was specific in the recognition of IBV antibodies. In addition, the data from the detection of field samples and IBV strains indicated that using the recombinant protein as coating antigen could achieve an equivalent performance to an ELISA kit based on infected material extracts as a source of antigen(s). ELISAs based on recombinant proteins are safe ( no live virus), clean ( only virus antigens are present), specific ( single proteins can be used) and rapid ( to respond to new viral strains and strains that cannot necessarily be easily cultured).

Evaluation of the time resolved fluorescence immunoassay (TRFIA) for the detection of varicella zoster virus (VZV) antibodies following vaccination of healthcare workers

Determination of varicella zoster virus (VZV) immunity in healthcare workers without a history of chickenpox is important for identifying those in need of vOka vaccination. Post immunisation, healthcare workers in the UK who work with high risk patients are tested for seroconversion. To assess the performance of the time-resolved fluorescence immunoassay (TRFIA) for the detection of antibody in vaccinated as well as unvaccinated individuals, a cut-off was first calculated. VZV-IgG specific avidity and titres six weeks after the first dose of vaccine were used to identify subjects with pre-existing immunity among a cohort of 110 healthcare workers. Those with high avidity (≥60%) were considered to have previous immunity to VZV and those with low or equivocal avidity (<60%) were considered naive. The former had antibody levels ≥400mIU/mL and latter had levels <400mIU/mL. Comparison of the baseline values of the naive and immune groups allowed the estimation of a TRFIA cut-off value of >130mIU/mL which best discriminated between the two groups and this was confirmed by ROC analysis. Using this value, the sensitivity and specificity of TRFIA cut-off were 90% (95% CI 79-96), and 78% (95% CI 61-90) respectively in this population. A subset of samples tested by the gold standard Fluorescence Antibody to Membrane Antigen (FAMA) test showed 84% (54/64) agreement with TRFIA.

Replication enhancer elements within the open reading frame of tick-borne encephalitis virus and their evolution within the Flavivirus genus

We provide experimental evidence of a replication enhancer element (REE) within the capsid gene of tick-borne encephalitis virus (TBEV, genus Flavivirus). Thermodynamic and phylogenetic analyses predicted that the REE folds as a long stable stem–loop (designated SL6), conserved among all tick-borne flaviviruses (TBFV). Homologous sequences and potential base pairing were found in the corresponding regions of mosquito-borne flaviviruses, but not in more genetically distant flaviviruses. To investigate the role of SL6, nucleotide substitutions were introduced which changed a conserved hexanucleotide motif, the conformation of the terminal loop and the base-paired dsRNA stacking. Substitutions were made within a TBEV reverse genetic system and recovered mutants were compared for plaque morphology, single-step replication kinetics and cytopathic effect. The greatest phenotypic changes were observed in mutants with a destabilized stem. Point mutations in the conserved hexanucleotide motif of the terminal loop caused moderate virus attenuation. However, all mutants eventually reached the titre of wild-type virus late post-infection. Thus, although not essential for growth in tissue culture, the SL6 REE acts to up-regulate virus replication. We hypothesize that this modulatory role may be important for TBEV survival in nature, where the virus circulates by non-viraemic transmission between infected and non-infected ticks, during co-feeding on local rodents.

Efficient production of foot-and-mouth disease virus empty capsids in insect cells following down regulation of 3C protease activity

Foot-and-mouth disease virus (FMDV) is a significant economically and distributed globally pathogen of Artiodactyla. Current vaccines are chemically inactivated whole virus particles that require large-scale virus growth in strict bio-containment with the associated risks of accidental release or incomplete inactivation. Non-infectious empty capsids are structural mimics of authentic particles with no associated risk and constitute an alternate vaccine candidate. Capsids self-assemble from the processed virus structural proteins, VP0, VP3 and VP1, which are released from the structural protein precursor P1-2A by the action of the virus-encoded 3C protease. To date recombinant empty capsid assembly has been limited by poor expression levels, restricting the development of empty capsids as a viable vaccine. Here expression of the FMDV structural protein precursor P1-2A in insect cells is shown to be efficient but linkage of the cognate 3C protease to the C-terminus reduces expression significantly. Inactivation of the 3C enzyme in a P1-2A-3C cassette allows expression and intermediate levels of 3C activity resulted in efficient processing of the P1-2A precursor into the structural proteins which assembled into empty capsids. Expression was independent of the insect host cell background and leads to capsids that are recognised as authentic by a range of anti-FMDV bovine sera suggesting their feasibility as an alternate vaccine.

Expression, purification and crystallization of the ectodomain of the envelope glycoprotein E2 from Bovine viral diarrhoea virus

Bovine viral diarrhoea virus (BVDV) is an economically important animal pathogen which is closely related to Hepatitis C virus. Of the structural proteins, the envelope glycoprotein E2 of BVDV is the major antigen which induces neutralizing antibodies; thus, BVDV E2 is considered as an ideal target for use in subunit vaccines. Here, the expression, purification of wild-type and mutant forms of the ectodomain of BVDV E2 and subsequent crystallization and data collection of two crystal forms grown at low and neutral pH are reported. Native and multiple-wavelength anomalous dispersion (MAD) data sets have been collected and structure determination is in progress.

Highly potent delivery method of gp160 envelope vaccine combining lentivirus-like particles and DNA electrotransfer

Particulate antigen assemblies in the nanometer range and DNA plasmids are particularly interesting for designing vaccines. We hypothesised that a combination of these approaches could result in a new delivery method of gp160 envelope HIV-1 vaccine which could combine the potency of virus-like particles (VLPs) and the simplicity of use of DNA vaccines. Characterisation of lentivirus-like particles (lentiVLPs) by western blot, dynamic light scattering and electron microscopy revealed that their protein pattern, size and structure make them promising candidates for HIV-1 vaccines. Although all particles were similar with regard to size and distribution, they clearly differed in p24 capsid protein content suggesting that Rev may be required for particle maturation and Gag processing. In vivo, lentiVLP pseudotyping with the gp160 envelope or with a combination of gp160 and VSV-G envelopes did not influence the magnitude of the immune response but the combination of lentiVLPs with Alum adjuvant resulted in a more potent response. Interestingly, the strongest immune response was obtained when plasmids encoding lentiVLPs were co-delivered to mice muscles by electrotransfer, suggesting that lentiVLPs were efficiently produced in vivo or the packaging genes mediate an adjuvant effect. DNA electrotransfer of plasmids encoding lentivirus-like particles offers many advantages and appears therefore as a promising delivery method of HIV-1 vaccines. Keywords:VLP, Electroporation, Electrotransfer, HIV vaccine, DNA vaccine

Structure-based energetics of protein interfaces guides foot-and-mouth disease virus vaccine design

Virus capsids are primed for disassembly, yet capsid integrity is key to generating a protective immune response. Foot-and-mouth disease virus (FMDV) capsids comprise identical pentameric protein subunits held together by tenuous noncovalent interactions and are often unstable. Chemically inactivated or recombinant empty capsids, which could form the basis of future vaccines, are even less stable than live virus. Here we devised a computational method to assess the relative stability of protein-protein interfaces and used it to design improved candidate vaccines for two poorly stable, but globally important, serotypes of FMDV: O and SAT2. We used a restrained molecular dynamics strategy to rank mutations predicted to strengthen the pentamer interfaces and applied the results to produce stabilized capsids. Structural analyses and stability assays confirmed the predictions, and vaccinated animals generated improved neutralizing-antibody responses to stabilized particles compared to parental viruses and wild-type capsids.