Rob Noad

Virus-like particles as immunogens

Subunit vaccines based on recombinant proteins can suffer from poor immunogenicity owing to incorrect folding of the target protein or poor presentation to the immune system. Virus-like particles (VLPs) represent a specific class of subunit vaccine that mimic the structure of authentic virus particles. They are recognized readily by the immune system and present viral antigens in a more authentic conformation than other subunit vaccines. VLPs have therefore shown dramatic effectiveness as candidate vaccines. Here, we review the current status of VLPs as vaccines, and discuss the characteristics and problems associated with producing VLPs for different viruses.

Virus-like particles as a vaccine delivery system: myths and facts.

Vaccines against viral disease have traditionally relied on attenuated virus strains or inactivation of infectious virus. Subunit vaccines based on viral proteins expressed in heterologous systems have been effective for some pathogens, but have often suffered from poor immunogenicity due to incorrect protein folding or modification. In this review we focus on a specific class of viral subunit vaccine that mimics the overall structure of virus particles and thus preserves the native antigenic conformation of the immunogenic proteins. These virus‑like particles (VLPs) have been produced for a wide range of taxonomically and structurally distinct viruses, and have unique advantages in terms of safety and immunogenicity over previous approaches. With new VLP vaccines for papillomavirus beginning to reach the market place we argue that this technology has now ‘come‑of‑age’ and must be considered a viable vaccine strategy.

Purified Recombinant Bluetongue Virus VP1 Exhibits RNA Replicase Activity

ABSTRACT The polymerase protein of all known double-stranded RNA (dsRNA) viruses is located within a complex subviral core particle that is responsible for transcription of the viral genome. For members of the family Reoviridae, this particle allows messenger sense RNA synthesis while sequestering the viral genome away from cellular dsRNA surveillance systems during infection of eukaryotic cells. The core particle of bluetongue virus (BTV) consists of the major structural proteins VP3 and VP7 and the minor enzymatic proteins VP1 (polymerase), VP4 (capping enzyme), and VP6 (helicase). In this report we have characterized fully processive dsRNA synthesis by VP1 from a viral plus-strand RNA template in the absence of the other proteins of the BTV core. This replicase activity consists of de novo initiation of synthesis, followed by elongation of the minus strand. Purified VP1 exhibits little sequence specificity for BTV plus-strand template, suggesting that the choice of viral over nonviral RNA template comes from its association with other proteins within the viral core.

Prospects for improved bluetongue vaccines.

Bluetongue has been recognized as a viral disease of livestock for more than 100 years. Repeated incursions of Bluetongue into Europe since 1998 have been particularly devastating for highly sensitive European fine-wool sheep breeds, and have resulted in a resurgence of interest in vaccine manufacture. Fortunately, the virus and its serology are well understood and vaccination prevents the disease. However, current vaccines are not without their problems, and many new approaches are being tested to improve the safety and breadth of protection afforded. This Review describes the leading technologies for improved bluetongue vaccines and looks ahead to how advances in other viral vaccines might be applied to this disease.

Interaction between Bluetongue virus outer capsid protein VP2 and vimentin is necessary for virus egress

BackgroundThe VP2 outer capsid protein Bluetongue Virus (BTV) is responsible for receptor binding, haemagglutination and eliciting host-specific immunity. However, the assembly of this outer capsid protein on the transcriptionally active viral core would block transcription of the virus. Thus assembly of the outer capsid on the core particle must be a tightly controlled process during virus maturation. Earlier studies have detected mature virus particles associated with intermediate filaments in virus infected cells but the viral determinant for this association and the effect of disrupting intermediate filaments on virus assembly and release are unknown.ResultsIn this study it is demonstrated that BTV VP2 associates with vimentin in both virus infected cells and in the absence of other viral proteins. Further, the determinants of vimentin localisation are mapped to the N-terminus of the protein and deletions of aminio acids between residues 65 and 114 are shown to disrupt VP2-vimentin association. Site directed mutation also reveals that amino acid residues Gly 70 and Val 72 are important in the VP2-vimentin association. Mutation of these amino acids resulted in a soluble VP2 capable of forming trimeric structures similar to unmodified protein that no longer associated with vimentin. Furthermore, pharmacological disruption of intermediate filaments, either directly or indirectly through the disruption of the microtubule network, inhibited virus release from BTV infected cells.ConclusionThe principal findings of the research are that the association of mature BTV particles with intermediate filaments are driven by the interaction of VP2 with vimentin and that this interaction contributes to virus egress. Furthermore, i) the N-terminal 118 amino acids of VP2 are sufficient to confer vimentin interaction. ii) Deletion of amino acids 65–114 or mutation of amino acids 70–72 to DVD abrogates vimentin association. iii) Finally, disruption of vimentin structures results in an increase in cell associated BTV and a reduction in the amount of released virus from infected cells.

Bluetongue Virus Outer Capsid Proteins Are Sufficient To Trigger Apoptosis in Mammalian Cells

ABSTRACT Bluetongue virus (BTV) is transmitted by Culicoides sp. biting midges to livestock, causing severe hemorrhagic disease in sheep, but is asymptomatic in the insect host. Similarly, BTV causes rapid cell death in infected mammalian cells in culture, whereas infections of insect cells are long-term and unapparent, despite productive virus replication. To assess whether apoptosis plays any role in these two distinct cell responses, we have investigated apoptosis in cultured insect and mammalian cells. Three different mammalian cell lines and three different insect cell lines including Culicoides variipennis (KC) cells were infected with BTV serotype 10, and the key apoptosis indicators of cell morphology, chromosomal DNA fragmentation, and caspase-3 activation were monitored. BTV infection induced apoptosis with the activation of the transcription factor nuclear factor κB (NF-κB) in all three mammalian cell lines. In contrast, no evidence for apoptosis was detected in any of the three insect cell lines in response to BTV infection. Using inhibitors of endosomal acidification and UV-inactivated virus, we established that virus uncoating, but not productive virus replication, is necessary for BTV to trigger apoptosis in mammalian cells. Intracellular expression of the viral outer capsid proteins VP2 and VP5 or the two major nonstructural proteins NS1 and NS2 was not sufficient to trigger an apoptotic response. However, extracellular treatment with a combination of purified recombinant VP2 and VP5, but not with each protein used separately, resulted in an apoptotic response. Virus- and VP2-VP5-stimulated apoptotic responses were both inhibited by inhibitors of endosomal acidification. Thus, for BTV the viral outer capsid proteins alone are sufficient to trigger apoptosis.

Characterisation of a GII-4 norovirus variant-specific surface-exposed site involved in antibody binding

BackgroundThe human noroviruses are a highly diverse group of viruses with a single-stranded RNA genome encoding a single major structural protein (VP1), which has a hypervariable domain (P2 domain) as the most exposed part of the virion. The noroviruses are classified on the basis of nucleotide sequence diversity in the VP1-encoding ORF2 gene, which divides the majority of human noroviruses into two genogroups (GI and GII). GII-4 noroviruses are the major aetiological agent of outbreaks of gastroenteritis around the world. During a winter season the diversity among the GII-4 noroviruses has been shown to fluctuate, driving the appearance of new virus variants in the population. We have previously shown that sequence data and in silico modelling experiments suggest there are two surface-exposed sites (site A and site B) in the hypervariable P2 domain. We predict these sites may form a functional variant-specific epitope that evolves under selective pressure from the host immune response and gives rise to antibody escape mutants.ResultsIn this paper, we describe the construction of recombinant baculoviruses to express VLPs representing one pre-epidemic and one epidemic variant of GII-4 noroviruses, and the production of monoclonal antibodies against them. We use these novel reagents to provide evidence that site A and site B form a conformational, variant-specific, surface-exposed site on the GII-4 norovirus capsid that is involved in antibody binding.ConclusionAs predicted by our earlier study, significant amino acid changes at site A and site B give rise to GII-4 norovirus epidemic variants that are antibody escape mutants.

Validation of a novel approach for the rapid production of immunogenic virus-like particles for bluetongue virus

Bluetongue virus causes an emerging disease of ruminants, principally affecting sheep and cattle. Since 1998, there have been multiple separate outbreaks of bluetongue disease in Europe that have highlighted the need for a safe, efficacious, DIVA compliant vaccine. We report here a new baculovirus expression strategy which allowed pre-integration of the genes encoding the BTV inner capsid proteins at one baculovirus locus and those encoding the outer capsid proteins at a different locus. A modified baculovirus with two marker proteins to facilitate the phenotypic selection of recombinant viruses was developed. The utility of this approach is demonstrated by the production of BTV VLPs to a number of serotypes. For a proof of concept, VLPs of one serotype was then tested for protective immune response. VLPs were demonstrated to be safe, highly effective, immunogens in sheep, reducing post-challenge viraemia to levels below the threshold detection limit of quantitative RT-PCR when vaccinated animals were challenged with virulent virus.

Multigene expression of protein complexes by iterative modification of genomic Bacmid DNA

BackgroundMany cellular multi-protein complexes are naturally present in cells at low abundance. Baculovirus expression offers one approach to produce milligram quantities of correctly folded and processed eukaryotic protein complexes. However, current strategies suffer from the need to produce large transfer vectors, and the use of repeated promoter sequences in baculovirus, which itself produces proteins that promote homologous recombination. One possible solution to these problems is to construct baculovirus genomes that express each protein in a complex from a separate locus within the viral DNA. However current methods for selecting such recombinant genomes are too inefficient to routinely modify the virus in this way.ResultsThis paper reports a method which combines the lambda red and bacteriophage P1 Cre-recombinase systems to efficiently generate baculoviruses in which protein complexes are expressed from multiple, single-locus insertions of foreign genes. This method is based on an 88 fold improvement in the selection of recombinant viruses generated by red recombination techniques through use of a bipartite selection cassette. Using this system, seven new genetic loci were identified in the AcMNPV genome suitable for the high level expression of recombinant proteins. These loci were used to allow the recovery two recombinant virus-like particles with potential biotechnological applications (influenza A virus HA/M1 particles and bluetongue virus VP2/VP3/VP5/VP7 particles) and the mammalian chaperone and cancer drug target CCT (16 subunits formed from 8 proteins).Conclusion1. Use of bipartite selections can significantly improve selection of modified bacterial artificial chromosomes carrying baculovirus DNA. Furthermore this approach is sufficiently robust to allow routine modification of the virus genome. 2. In addition to the commonly used p10 and polyhedrin loci, the ctx, egt, 39k, orf51, gp37, iap2 and odv-e56 loci in AcMNPV are all suitable for the high level expression of heterologous genes. 3. Two protein, four protein and eight protein complexes including virus-like particles and cellular chaperone complexes can be produced using the new approach.

Rehosting of Bacterial Chaperones for High-Quality Protein Production

ABSTRACT Coproduction of DnaK/DnaJ in Escherichia coli enhances solubility but promotes proteolytic degradation of their substrates, minimizing the yield of unstable polypeptides. Higher eukaryotes have orthologs of DnaK/DnaJ but lack the linked bacterial proteolytic system. By coexpression of DnaK and DnaJ in insect cells with inherently misfolding-prone recombinant proteins, we demonstrate simultaneous improvement of soluble protein yield and quality and proteolytic stability. Thus, undesired side effects of bacterial folding modulators can be avoided by appropriate rehosting in heterologous cell expression systems.