Fuxiao Liu

Use of baculovirus expression system for generation of virus-like particles: Successes and challenges

Abstract The baculovirus expression system (BES) has been one of the versatile platforms for the production of recombinant proteins requiring multiple post-translational modifications, such as folding, oligomerization, phosphorylation, glycosylation, acylation, disulfide bond formation and proteolytic cleavage. Advances in recombinant DNA technology have facilitated application of the BES, and made it possible to express multiple proteins simultaneously in a single infection and to produce multimeric proteins sharing functional similarity with their natural analogs. Therefore, the BES has been used for the production of recombinant proteins and the construction of virus-like particles (VLPs), as well as for the development of subunit vaccines, including VLP-based vaccines. The VLP, which consists of one or more structural proteins but no viral genome, resembles the authentic virion but cannot replicate in cells. The high-quality recombinant protein expression and post-translational modifications obtained with the BES, along with its capacity to produce multiple proteins, imply that it is ideally suited to VLP production. In this article, we critically review the pros and cons of using the BES as a platform to produce both enveloped and non-enveloped VLPs.

Virus-like particles: potential veterinary vaccine immunogens.

Virus-like particle (VLP) composed of outer shell but no genome of virus mimics the natural configuration of authentic virion and has no characteristics of self-replication. A close resemblance to native viruses in molecular scaffolds and an absence of genomes make VLPs effectively elicit both humoral and cell-mediated immune responses even with no requirement of adjuvant for vaccines. As effective immunogens, characterized by high immunogenicity and safety, VLPs have been employed in production of human vaccines, such as the licensed vaccines of hepatitis B virus and human papillomavirus. However, there has been no report of licensed veterinary VLP vaccine worldwide as yet. Despite the wide application in vaccination, both the conventional inactivated and live attenuated vaccines for animals are subject to potential limitations due to incomplete inactivation and reversion to virulence. Therefore, those conventional vaccines may, to some extent, be replaced with the VLP-based vaccines conferring higher protection and safety to vaccinated animals. Here, we review the current status of VLPs as veterinary vaccines, and discuss the characteristics and problems associated with generating VLPs for different animal viruses.

Expression, purification and characterization of two truncated peste des petits ruminants virus matrix proteins in Escherichia coli, and production of polyclonal antibodies against this protein.

Peste des petits ruminants virus (PPRV), the etiological agent of peste des petits ruminants, is classified into the genus Morbillivirus in the family Paramyxoviridae. The PPRV matrix (M) gene is composed of 1483 base pairs, encoding a 335 amino acids M protein with a molecular weight of approximately 38kD. We have demonstrated previously that the full-length M protein was expressed at an extremely low level or not even expressed in Escherichia coli BL21 (DE3). In this study, the M protein was split into two truncated forms to be successfully expressed in E. coli at a high level using the pET30a (+) vector, respectively, by analysis of SDS-PAGE, western blot and MALDI-TOF-MS. The optimization of culture conditions led us to perform the recombinant protein induction with 0.2mM IPTG at 28°C for 12h, whereby both proteins nevertheless were expressed in the insoluble form. Therefore, both His-tagged proteins were purified under the denaturing condition using a commercially available kit. Balb/c mice were immunized with the complex of purified proteins and then effectively produced polyclonal antibodies, which reached to a relatively high titer by the analysis of ELISA. The specificity of the prepared polyclonal antibodies was checked by western blot and immunofluorescence, revealing them with the desirable specificity against both non-denatured and denatured M proteins.

Virus-like particles: promising platforms with characteristics of DIVA for veterinary vaccine design.

In general, it is difficult to differentiate infected from vaccinated animals through vaccination with conventional vaccines, thereby impeding the serological surveillance of animal diseases. DIVA (differentiating infected from vaccinated animals) vaccine, originally known as marker vaccine, usually based on the absence of at least one immunogenic protein in the vaccine strain, allows DIVA in conjunction with a diagnostic test that detects antibodies against the antigens lacking in the vaccine strain. Virus-like particles (VLPs), composed of one or more structural proteins but no genomes of native viruses, mimic the organization and conformation of authentic virions but have no ability to self-replicate in cells, potentially yielding safer vaccine candidates. Since VLPs containing either monovalent or multivalent antigen can be produced in compliance with the requirements for serological surveillance, the use of VLP-based vaccines plays a promising role in DIVA vaccination strategies against animal diseases. Here, we critically reviewed VLPs and companion diagnostics with properties of DIVA for veterinary vaccine design, and three different VLPs as promising platforms for DIVA vaccination strategies in animals.

Formation of peste des petits ruminants spikeless virus-like particles by co-expression of M and N proteins in insect cells.

Peste des petits ruminants virus (PPRV) has a non-segmented negative sense RNA genome and is classified within the Morbillivirus genus of the Paramyxoviridae. Using the Bac-to-Bac® baculovirus expression system, we constructed recombinant baculoviruses that were able to co-express the PPRV matrix and nucleocapsid proteins in insect cells under the control of the polyhedron and p10 promoters, respectively. The results showed that although both structural proteins were expressed at a relatively low level, the interaction between them caused the formation of virus-like particles (VLPs) by viewing of transmission electron microscopy. The VLPs morphologically resembled authentic PPRVs but lacked spikes protruding from the particulate surfaces. Interestingly, the diameter of PPRV VLPs ranged from 100 to 150 nm, far less than the mean diameter (400-500 nm) of parental virions.

Current perspectives on conventional and novel vaccines against peste des petits ruminants

Peste des petits ruminants (PPR) is an acute or subacute, highly contagious viral disease of small ruminants, characterized by fever, oculonasal discharges, stomatitis, diarrhoea and pneumonia. This disease is included in the OIE (Office International des Epizooties) list of notifiable terrestrial animal diseases. PPR was first described in the early 1940s in Côte d′Ivoire, and at present, PPR is mainly circulating in Western and Central Africa, the Arabian Peninsula and Southern Asia. Peste des petits ruminants virus (PPRV), the etiological agent of PPR, is classified into the genus Morbillivirus in the family Paramyxoviridae, as its biological and physicochemical features are closely related to the other morbilliviruses. The first homologous PPR vaccine was developed by an artificially attenuated PPRV, named as Nigeria 75/1, which has been widely used in the production of live attenuated vaccines to protect small ruminants. A new generation of PPR vaccine candidates can be genetically modified to differentiate infected from vaccinated animals (DIVA), which nevertheless is difficult to achieve by conventional vaccines. In this review, we systematically discussed a broad range of vaccines against PPR, including commercially available vaccines and potential vaccine candidates, and further DIVA strategies for immunization with the new generation vaccines.

Budding of peste des petits ruminants virus-like particles from insect cell membrane based on intracellular co-expression of peste des petits ruminants virus M, H and N proteins by recombinant baculoviruses

Peste des petits ruminants virus (PPRV), an etiological agent of peste des petits ruminants (PPR), is classified into the genus Morbillivirus in the family Paramyxovirida. In this study, two full-length open reading frames (ORF) corresponding to the PPRV matrix (M) and haemagglutinin (H) genes underwent a codon-optimization based on insect cells, respectively. Two codon-optimized ORFs along with one native nucleocapsid (N) ORF were used to construct recombinant baculoviruses co-expressing the PPRV M, H and N proteins in insect cells. Analysis of Western blot, immunofluorescence, confocal microscopy and flow cytometry demonstrated co-expression of the three proteins but at different levels in insect cells, and PPR virus-like particles (VLPs) budded further from cell membrane based on self-assembly of the three proteins by viewing of ultrathin section with a transmission electron microscope (TEM). Subsequently, a small number of VLPs were purified by sucrose density gradient centrifugation for TEM viewing. The PPR VLPs, either purified by sucrose density gradient centrifugation or budding from insect cell membrane on ultrathin section, morphologically resembled authentic PPRVs but were smaller in diameter by the TEM examination.

Peste des petits ruminants virus-like particles induce both complete virus-specific antibodies and virus neutralizing antibodies in mice.

Peste des petits ruminants virus (PPRV), an etiological agent of peste des petits ruminants (PPR), is classified into the genus Morbillivirus in the family Paramyxoviridae. In a previous study, a recombinant baculovirus has been constructed to co-express the PPRV matrix (M), haemagglutinin (H) and nucleocapsid (N) proteins in insect cells, causing budding of PPR virus-like particles (VLPs) from insect cell membranes by viewing of ultrathin section with a transmission electron microscope. In this follow-up study, these PPR VLPs were purified by sucrose density gradient centrifugation for immunizing mice twice. Three weeks post-primary immunization and 2 weeks post-secondary immunization, all serum samples were obtained and subsequently subjected to indirect ELISA detection on complete virus-specific antibodies. In addition, all serum samples, which were collected 2 weeks post-secondary immunization, were used for virus neutralization test on PPRV neutralizing antibodies. The results showed that the purified PPR VLPs induced both types of antibodies mentioned above in mice, indicating a given potential of VLP-based vaccine candidate against PPR.

Major mutation events in structural genes of peste des petits ruminants virus through serial passages in vitro

Peste des petits ruminants (PPR) is an highly contagious disease of small ruminants, and caused by peste des petits ruminants virus (PPRV), a member of the genus Morbillivirus in the family Paramyxoviridae. The first outbreak of PPR in China was officially reported in July 2007, when a PPRV strain was successfully isolated from a sick goat in Tibet, followed by sequencing at a full-genome level (China/Tibet/Geg/07-30, GenBank: FJ905304.1). To date, this isolate has been virulently attenuated by more than 90 serial passages in Vero-Dog-SLAM cells at our laboratory. In this study, a total of nine strains by serial passages (namely the 10th, 20th, 30th, 40th, 50th, 60th, 70th, 80th, and 90th passages) were chosen for sequencing of six structural genes in PPRV. The sequence analysis showed that mutation rates in all viral genes were relatively low, and only a few identical mutations within certain genes were stably maintained after an earlier passage, perhaps indicating a predominance of mutants after such a passage.

Small interfering RNAs targeting peste des petits ruminants virus M mRNA increase virus-mediated fusogenicity and inhibit viral replication in vitro

Peste des petits ruminants (PPR), caused by peste des petits ruminants virus (PPRV), is an acute or subacute, highly contagious and economically important disease of small ruminants. The PPRV is classified into the genus Morbillivirus in the family Paramyxoviridae. The PPRV matrix (M) protein possesses an intrinsic ability to bind to lipid membranes, and plays a crucial role in viral assembly and further budding. In this study, three different small interfering RNAs (siRNA) were designed on the basis of translated region for PPRV Nigeria 75/1M mRNA, and were subsequently synthesized for their transfection into Vero-SLAM cells, followed by infection with PPRVs. The results showed that two out of three siRNAs robustly induced cell-to-cell fusion as early as 36h post-infection with PPRVs, effectively suppressed expression of the M protein by interference for the M mRNA, and eventually inhibited viral replication in vitro. These findings led us to speculate that siRNA-mediated knockdown of the M protein would alter its interaction with viral glycoproteins, thus exacerbating intercellular fusion but hampering virus release.