Towards a unique and transmissible vaccine against myxomatosis and rabbit haemorrhagic disease for rabbit populations

Currently available vaccines against myxomatosis and rabbit hemorrhagic disease virus (RHDV) are not suited to immunise wild rabbit populations, as vaccines need to be delivered individually by conventional veterinary practices. As an alternative approach, research in Spain has focused on the development of a transmissible vaccine. A recombinant virus has been constructed based on a naturally attenuated myxoma virus (MV) field strain, expressing the RHDV capsid protein (VP60). Following inoculation of rabbits, the recombinant virus (MV-VP60) induced specific antibody responses against MV and RHDV, conferring protection against lethal challenges with both viruses. Furthermore, the recombinant MV-VP60 virus showed a limited horizontal transmission capacity, either by direct contact or in a flea-mediated process, promoting immunisation of contact uninoculated animals. Efficacy and safety of the vaccine have been extensively evaluated under laboratory conditions and in a limited field trial. The development of the transmissible vaccine strategy and the steps being taken to obtain the marketing authorisation for the vaccine in the European Union are presented in this review. © CSIRO 2007

Recombinant Swinepox virus expressing β-galactosidase Investigation of vital host range and gene expression levels in cell culture

Swinepox virus (SPV) has been proposed as a potential vector for generating recombinant vaccines for swine. However, little is known about important aspects of SPV biology, such as the functionality of SPV promoters or the host range of SPV. Using a transient expression assay, well- characterized vaccinia virus promoters were shown to be active in cells infected with SPV. A recombinant SPV expressing β-galactosidase (β-gal) was constructed and characterized. The E. coli LacZ gene was placed under the control of a strong vaccinia synthetic early/late promoter and was inserted by homologous recombination in a noncoding region of the SPV genome. The recombinant SPV expressing β-gal was used to characterize the host range of the virus by measuring protein expression and virus production in different cell lines. In general, SPV expressed more protein and grew more efficiently than vaccinia virus in porcine cell lines. Surprisingly, the recombinant SPV was able to infect and replicate in several cell lines of nonswine origin. The virus directed regulated early and late gene expression of β-gal in those cells and formed blue plaques in cell monolayers in the presence of X- gal. Upon infection with the recombinant SPV, there was a significant level of viral replication, and the virus can be serially passaged in some nonswine cell lines. The data presented suggest that despite the strict host tropism of SPV, the virus exhibits a relatively broad host range in cell culture

Design of novel vaccines based on virus-like particles or chimeric virions

Virus-like particles (VLPs) are formed by viral structural proteins that, when overexpressed, spontaneously self-assemble into particles that are antigenically indistinguishable from infectious virus or subviral particles. VLPs are appealing as vaccine candidates because their inherent properties (i.e.;virus-sized, multimeric antigens, highly organised and repetitive structure, not infectious) are suitable for the induction of safe and efficient humoral and cellular immune responses. VLP-based vaccines have already been licensed for human and veterinary use, and many more vaccine candidates are currently in late stages of evaluation. Moreover, the development of VLPs as platforms for foreign antigen display has further broadened their potential applicability both as prophylactic and therapeutic vaccines. This chapter provides an overview on the design and use of VLPs for the development of new generation vaccines

Virus-like particle-based vaccines for animal viral infections

Vaccination is considered one of the most effective ways to control pathogens and prevent diseases in humans as well as in the veterinary field. Traditional vaccines against animal viral diseases are based on inactivated or attenuated viruses, but new subunit vaccines are gaining attention from researchers in animal vaccinology. Among these, virus-like particles (VLPs) represent one of the most appealing approaches opening up interesting frontiers in animal vaccines. VLPs are robust protein scaffolds exhibiting well-defined geometry and uniformity that mimic the overall structure of the native virions but lack the viral genome. They are often antigenically indistinguishable from the virus from which they were derived and present important advantages in terms of safety. VLPs can stimulate strong humoral and cellular immune responses and have been shown to exhibit self-adjuvanting abilities. In addition to their suitability as a vaccine for the homologous virus from which they are derived, VLPs can also be used as vectors for the multimeric presentation of foreign antigens. VLPs have therefore shown dramatic effectiveness as candidate vaccines; nevertheless, only one veterinary VLP-base vaccine is licensed. Here, we review and examine in detail the current status of VLPs as a vaccine strategy in the veterinary field, and discuss the potential advantages and challenges of this technology. © 2012 Sociedad Española de Inmunología. Published by Elsevier España, S.L. All rights reserved

Virus-like particles The new frontier of vaccines for animal viral infections

Vaccination continues to be the main approach to protect animals from infectious diseases. Until recently, all licensed vaccines were developed using conventional technologies. Subunit vaccines are, however, gaining attention from researchers in the field of veterinary vaccinology, and among these, virus-like particles (VLPs) represent one of the most appealing approaches. VLPs are robust protein cages in the nanometer range that mimic the overall structure of the native virions but lack the viral genome. They are often antigenically indistinguishable from the virus from which they were derived and present important advantages in terms of safety. VLPs can stimulate strong humoral and cellular immune responses and have been shown to exhibit self-adjuvanting abilities. In addition to their suitability as a vaccine for the homologous virus from which they are derived, VLPs can also be used as vectors for the multimeric presentation of foreign antigens. VLPs have therefore shown dramatic effectiveness as candidate vaccines. Here, we review the current status of VLPs as a vaccine technology in the veterinary field, and discuss the potential advantages and challenges of this technology. © 2012 Elsevier B.V

Sequence and analysis of a swinepox virus homologue of the vaccinia virus major envelope protein P37 (F13L)

P37 (F13L gene product), the most abundant protein in the envelope of the extracellular virus form of the prototype poxvirus, vaccinia virus (VV), is a crucial player in the process leading to acquisition of the envelope, virus egress and transmission. We have cloned and sequenced a swinepox virus (SPV) gene homologous to VV F13L. The SPV gene product, termed P42, was 54% identical to P37, the VV F13L gene product, and, among the poxviruses, was most similar (73% identity) to the myxoma virus homologue. The SPV P42 gene contained late transcription signals and was expressed only at late times during infection. The protein was palmitylated, and showed an intracellular distribution similar to that of VV P37, both by immunofluorescence and by subcellular fractionation. As with VV P37, SPV P42 was incorporated in extracellular enveloped SPV particles, but was absent from the intracellular mature virus form. To check the ability of SPV P42 to function in the context of VV infection, we inserted the SPV gene into a VV deficient in P37, which is severely blocked in virus envelopment and cell-to-cell transmission. Despite correct expression of SPV P42, the resulting recombinant VV showed no rescue of extracellular virus formation or cell-to-cell virus spread. The lack of function of SPV P42 in the VV genetic background suggests that specific interactions between SPV P42 or VV P37 and other viral proteins is required to drive the envelopment process

Synthesis in vitro of rabbit hemorrhagic disease virus subgenomic RNA by internal initiation on (-)sense genomic RNA Mapping of a subgenomic promoter

Rabbit hemorrhagic disease virus (RHDV), a positive-strand RNA virus, is the type species of the Lagovirus within the Caliciviridae. In addition to the genomic RNA of 7.4 kb, a subgenomic mRNA (sgRNA) of 2.2 kb, which is identical in sequence to the 3′ one-third of the genomic RNA, is also synthesized in RHDV-infected cells. Numerous RNA viruses make sgRNA for expression of their 3′-proximal genes. A relevant mechanism for viral gene expression is the regulation of sgRNA synthesis by specific promoter elements. In this study, we have investigated in vitro the sgRNA synthesis mechanism using recombinant RHDV RNA-dependent RNA polymerase produced in baculovirus-infected insect cells and synthetic RHDV (-)RNAs of different lengths containing regions located upstream of the subgenomic start site. We report evidences supporting that the sgRNA of RHDV is synthesized in vitro by internal initiation (subgenomic promoter) on (-)RNA templates of genomic length. The deletion mapping of the subgenomic promoter starting from minus-strand genomic length RNA showed that a sequence of 50 nucleotides upstream of the sgRNA start site (+1) is sufficient for full subgenomic promoter activity in an in vitro assay using recombinant RHDV RNA-dependent RNA polymerase. This study reports the first description of a subgenomic promoter in a member of the Caliciviridae

Development of a low-cost, insect larvae-derived recombinant subunit vaccine against RHDV

Vaccine antigens against rabbit hemorrhagic disease virus (RHDV) are currently derived from inactivated RHDV obtained from livers of experimentally infected rabbits. Several RHDV-derived recombinant immunogens have been reported. However, their application in vaccines has been restricted due to their high production costs. In this paper, we describe the development of an inexpensive, safe, stable vaccine antigen for RHDV. A baculovirus expressing a recombinant RHDV capsid protein (VP60r) was used to infect Trichoplusia ni insect larvae. It reached an expression efficiency of 12.5% of total soluble protein, i.e. ∼ 2 mg of VP60r per larva. Preservation of the antigenicity and immunogenicity of the VP60r was confirmed by immunological and immunization experiments. Lyophilized crude larvae extracts, containing VP60r, were stable, at room temperature, for at least 800 days. In all cases, rabbits immunized with a single dose of VP60r by the intramuscular route were protected against RHDV challenge. Doses used were as low as 2 μg of VP60r in the presence of adjuvant or 100 μg without one. Orally administered VP60r in the absence of an adjuvant gave no protection. The potential costs of an RHDV vaccine made using this technology would be reduced considerably compared with producing the same protein in insect cells maintained by fermentation. In conclusion, the larva expression system may provide a broad-based strategy for production of recombinant subunit antigens (insectigens) for human or animal medicines, especially when production costs restrain their use. © 2007 Elsevier Inc. All rights reserved

Improved production efficiency of virus-like particles by the baculovirus expression vector system

Vaccines based on virus-like particles (VLPs) have proven effective in humans and animals. In this regard, the baculovirus expression vector system (BEVS) is one of the technologies of choice to generate such highly immunogenic vaccines. The extended use of these vaccines for human and animal populations is constrained because of high production costs, therefore a significant improvement in productivity is crucial to ensure their commercial viability. Here we describe the use of the previously described baculovirus expression cassette, called TB, to model the production of two VLP-forming vaccine antigens in insect cells. Capsid proteins from porcine circovirus type 2 (PCV2 Cap) and from the calicivirus that causes rabbit hemorrhagic disease (RHDV VP60) were expressed in insect cells using baculoviruses genetically engineered with the TB expression cassette. Productivity was compared to that obtained using standard counterpart vectors expressing the same proteins under the control of the polyhedrin promoter. Our results demonstrate that the use of the TB expression cassette increased the production yields of these vaccine antigens by around 300% with respect to the standard vectors. The recombinant proteins produced by TB-modified vectors were fully functional, forming VLPs identical in size and shape to those generated by the standard baculoviruses, as determined by electron microscopy analysis. The use of the TB expression cassette implies a simple modification of the baculovirus vectors that significantly improves the cost efficiency of VLP-based vaccine production, thereby facilitating the commercial viability and broad application of these vaccines for human and animal health. © 2015 López-Vidal et al.This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited