A protocol for the gentle purification of virus-like particles produced in plants.

The purpose of the protocol is to extract and purify virus-like particles (VLPs) that have been produced in plants. More specifically, this method is well suited to the purification of chimaeric and genetically modified VLPs that do not have native surface properties. This will be the case for VLPs used in antigen display experiments. Such particles are often more fragile than their wild-type infectious virus counterparts, and as such can be damaged or lost during procedures that involve pelleting or precipitating the particles. The method presented here is based on ultracentrifugation and density gradients, with no pelleting or precipitation step. It makes virtually no assumptions about the yield of recombinant VLPs or their properties, which means that this protocol is ideally suited to screening new constructs which are expected to lead to the formation of VLPs. This protocol will allow the researcher to determine whether the construct does indeed form VLPs, and if it does, will reduce the likelihood of those particles being lost or damaged during the purification process. Because of its non-specific nature, this protocol may also be suited to the purification of viruses of unknown nature from leaf material where an infection is suspected.

Production and characterization of mammalian virus-like particles from modified vaccinia virus Ankara vectors expressing influenza H5N1 hemagglutinin and neuraminidase.

Several studies have described the production of influenza virus-like particles (VLP) using a variety of platform systems. These VLPs are non-replicating particles that spontaneously self-assemble from expressed influenza virus proteins and have been proposed as vaccine candidates for both seasonal and pandemic influenza. Although still in the early stages of development and evaluation as influenza vaccines, influenza VLPs have a variety of other valuable uses such as examining and understanding correlates of protection against influenza and investigating virus-cell interactions. The most common production system for influenza VLPs is the baculovirus-insect cell expression which has several attractive features including the ease in which new gene combinations can be constructed, the immunogenicity elicited and protection afforded by the produced VLPs, and the scalability offered by the system. However, there are differences between the influenza VLPs produced by baculovirus expression systems in insect cells and the influenza viruses produced for use as current vaccines or the virus produced during a productive clinical infection. We describe here the development of a modified vaccinia virus Ankara (MVA) system to generate mammalian influenza VLPs containing influenza H5N1 proteins. The MVA vector system is flexible for manipulating and generating various VLP constructs, expresses high level of influenza hemagglutinin (HA), neuraminidase (NA), and matrix (M) proteins, and can be scaled up to produce VLPs in quantities sufficient for in vivo studies. We show that mammalian VLPs are generated from recombinant MVA vectors expressing H5N1 HA alone, but that increased VLP production can be achieved if NA is co-expressed. These mammalian H5N1 influenza VLPs have properties in common with live virus, as shown by electron microscopy analysis, their ability to hemagglutinate red blood cells, express neuraminidase activity, and to bind influenza specific antibodies. Importantly, these VLPs are able to elicit a protective immune response in a mouse challenge model, suggesting their utility in dissecting the correlates of immunity in such models. Mammalian derived VLPs may also provide a useful tool for studying virus-cell interactions and may have potential for development as pandemic vaccines.

Bioprocessing of plant-derived virus-like particles of Norwalk virus capsid protein under current Good Manufacture Practice regulations.

Despite the success in expressing a variety of subunit vaccine proteins in plants and the recent stride in improving vaccine accumulation levels by transient expression systems, there is still no plant-derived vaccine that has been licensed for human use. The lack of commercial success of plant-made vaccines lies in several technical and regulatory barriers that remain to be overcome. These challenges include the lack of scalable downstream processing procedures, the uncertainty of regulatory compliance of production processes, and the lack of demonstration of plant-derived products that meet the required standards of regulatory agencies in identity, purity, potency and safety. In this study, we addressed these remaining challenges and successfully demonstrate the ability of using plants to produce a pharmaceutical grade Norwalk virus (NV) vaccine under current Good Manufacture Practice (cGMP) guidelines at multiple gram scales. Our results demonstrate that an efficient and scalable extraction and purification scheme can be established for processing virus-like particles (VLPs) of NV capsid protein (NVCP). We successfully operated the upstream and downstream NVCP production processes under cGMP regulations. Furthermore, plant-derived NVCP VLP demonstrates the identity, purity, potency and safety that meet the preset release specifications. This material is being tested in a Phase I human clinical trial. This research provides the first report of producing a plant-derived vaccine at scale under cGMP regulations in an academic setting and an important step for plant-produced vaccines to become a commercial reality.

Formation of virus-like particles from O-type foot-and-mouth disease virus in insect cells using codon-optimized synthetic genes.

A recombinant baculovirus was constructed to simultaneously express codon-optimized virus-like particles (VLP), A VP1-2A-VP3 and VP0 of serotype O foot-and-mouth disease virus (FMDV), from individual promoters. The target proteins were expressed in insect cells at high level, as shown by indirect sandwich ELISA; and the expressed VP1-2A-VP3 could autocatalytically be cleaved into the individual proteins, VP1-2A and VP3, as shown by Western-blot analyses. In addition, in the insect cells, the structural proteins, VP0, VP3 and VP1-2A, self-assembled into virus-like particles resembling the authentic FMDV particles. This information should prove useful for the development of more efficient VLP assembly using shorter genes.

Characterization and diagnostic potential of foreign epitope-presenting Ty1 virus-like particles expressed in Escherichia coli and Pichia pastoris.

We expressed a truncated p1 protein (p1-379) from the Saccharomyces cerevisiae retrotransposon Ty1 in the cytosol of Escherichia coli and Pichia pastoris, achieving maximum expression levels of 20 and 65 mg/l, respectively. Two well-characterized epitopes from beet necrotic yellow vein virus (BNYVV) were used to evaluate the virus-like particles (VLPs) as a presentation system for synthetic antigens. The epitopes were placed near the externally located N-terminus and at the internally located C-terminus of the p1 protein. Electron microscopy showed all particles to be morphologically similar to wild-type Ty1-VLPs from S. cerevisiae. However, fewer VLPs were observed in P. pastoris, suggesting that posttranslational modifications might inhibit binding to the carbon-coated electron microscopy grids. BNYVV epitopes were detected with specific monoclonal antibodies by Western blot and ELISA, with a detection limit as low as 5 ng/ml. The VLPs are therefore promising candidates for diagnostic standards and future vaccine development.

Morphogenesis of hepatitis B virus and its subviral envelope particles.

After cell hijacking and intracellular amplification, non-lytic enveloped viruses are usually released from the infected cell by budding across internal membranes or through the plasma membrane. The enveloped human hepatitis B virus (HBV) is an example of virus using an intracellular compartment to form new virions. Four decades after its discovery, HBV is still the primary cause of death by cancer due to a viral infection worldwide. Despite numerous studies on HBV genome replication little is known about its morphogenesis process. In addition to viral neogenesis, the HBV envelope proteins have the capability without any other viral component to form empty subviral envelope particles (SVPs), which are secreted into the blood of infected patients. A better knowledge of this process may be critical for future antiviral strategies. Previous studies have speculated that the morphogenesis of HBV and its SVPs occur through the same mechanisms. However, recent data clearly suggest that two different processes, including constitutive Golgi pathway or cellular machinery that generates internal vesicles of multivesicular bodies (MVB), independently form these two viral entities.

Japanese encephalitis virus-based replicon RNAs/particles as an expression system for HIV-1 Pr55 Gag that is capable of producing virus-like particles.

Ectopic expression of the structural protein Pr55(Gag) of HIV-1 has been limited by the presence of inhibitory sequences in the gag coding region that must normally be counteracted by HIV-1 Rev and RRE. Here, we describe a cytoplasmic RNA replicon based on the RNA genome of Japanese encephalitis virus (JEV) that is capable of expressing HIV-1 gag without requiring Rev/RRE. This replicon system was constructed by deleting all three JEV structural protein-coding regions (C, prM, and E) from the 5'-proximal region of the genome and simultaneously inserting an HIV-1 gag expression cassette driven by the internal ribosome entry site of encephalomyocarditis virus into the 3'-proximal noncoding region of the genome. Transfection of this JEV replicon RNA led to expression of Pr55(Gag) in the absence of Rev/RRE in the cytoplasm of hamster BHK-21, human HeLa, and mouse NIH/3T3 cells. Production of the Pr55(Gag) derived from this JEV replicon RNA appeared to be increased by approximately 3-fold when compared to that based on an alphavirus replicon RNA. Biochemical and morphological analyses demonstrated that the Pr55(Gag) proteins were released into the culture medium in the form of virus-like particles. We also observed that the JEV replicon RNAs expressing the Pr55(Gag) could be encapsidated into single-round infectious JEV replicon particles when transfected into a stable packaging cell line that provided the three JEV structural proteins in trans. This ectopic expression of the HIV-1 Pr55(Gag) by JEV-based replicon RNAs/particles in diverse cell types may represent a useful molecular platform for various biological applications in medicine and industry.

A DNA replicon system for rapid high-level production of virus-like particles in plants.

Recombinant virus-like particles (VLPs) represent a safe and effective vaccine strategy. We previously described a stable transgenic plant system for inexpensive production and oral delivery of VLP vaccines. However, the relatively low-level antigen accumulation and long-time frame to produce transgenic plants are the two major roadblocks in the practical development of plant-based VLP production. In this article, we describe the optimization of geminivirus-derived DNA replicon vectors for rapid, high-yield plant-based production of VLPs. Co-delivery of bean yellow dwarf virus (BeYDV)-derived vector and Rep/RepA-supplying vector by agroinfiltration of Nicotiana benthamiana leaves resulted in efficient replicon amplification and robust protein production within 5 days. Co-expression of the P19 protein of tomato bush stunt virus, a gene silencing inhibitor, further enhanced VLP accumulation by stabilizing the mRNA. With this system, hepatitis B core antigen (HBc) and Norwalk virus capsid protein (NVCP) were produced at 0.80 and 0.34 mg/g leaf fresh weight, respectively. Sedimentation analysis and electron microscopy of transiently expressed antigens verified the efficient assembly of VLPs. Furthermore, a single replicon vector containing a built-in Rep/RepA cassette without P19 drove protein expression at similar levels as the three-component system. These results demonstrate the advantages of fast and high-level production of VLP-based vaccines using the BeYDV-derived DNA replicon system for transient expression in plants.

Nef modulates the immunogenicity of Gag encoded in a non-infectious HIV DNA vaccine.

Gag-CD8+ T cell responses are associated with immune control of HIV infection. Since during HIV infection Nef impairs T cell responses, we evaluated whether deletion of nef from a non-infectious HIV DNA vaccine (Delta4 Nef+), creating Delta5 Nef(-), would affect its immunogenicity. When compared with Delta4, mice injected with Delta5 developed significantly lower CD8+ T cell responses to Gag, but no significant change in the responses to Env was observed. In vitro, deletion of Nef abrogated the induced cell death, production of virus-like particles and release of Gag from transfected cells. Thus, the effect of Nef in causing extrusion of Gag might adjuvant the CD8+ T cell responses to Gag in DNA vaccine.

The creation of stable cell lines expressing Ebola virus glycoproteins and the matrix protein VP40 and generating Ebola virus-like particles utilizing an ecdysone inducible mammalian expression system.

Ebola virus is a filovirus that causes hemorrhagic fever in humans and is associated with case fatality rates of up to 90%. The lack of therapeutic interventions in combination with the threat of weaponizing this organism has enhanced research investigations. The expression of key viral proteins and the production of virus-like particles in mammalian systems are often pursued for characterization and functional studies. Common practice is to express these proteins through transient transfection of mammalian cells. Unfortunately the transfection reagents are expensive and the process is time consuming and labour intensive. This work describes utilizing an ecdysone inducible mammalian expression system to create stable cell lines that express the Ebola virus transmembrane glycoprotein (GP), the soluble glycoprotein (sGP) and the matrix protein (VP40) individually as well as GP and VP40 simultaneously (for the production of virus like particles). These products were the same as those expressed by the transient system, by Western blot analysis and electron microscopy. The inducible system proved to be an improvement of the current technology by enhancing the cost effectiveness and simplifying the process.

RNase-resistant virus-like particles containing long chimeric RNA sequences produced by two-plasmid coexpression system.

RNase-resistant, noninfectious virus-like particles containing exogenous RNA sequences (armored RNA) are good candidates as RNA controls and standards in RNA virus detection. However, the length of RNA packaged in the virus-like particles with high efficiency is usually less than 500 bases. In this study, we describe a method for producing armored L-RNA. Armored L-RNA is a complex of MS2 bacteriophage coat protein and RNA produced in Escherichia coli by the induction of a two-plasmid coexpression system in which the coat protein and maturase are expressed from one plasmid and the target RNA sequence with modified MS2 stem-loop (pac site) is transcribed from another plasmid. A 3V armored L-RNA of 2,248 bases containing six gene fragments-hepatitis C virus, severe acute respiratory syndrome coronavirus (SARS-CoV1, SARS-CoV2, and SARS-CoV3), avian influenza virus matrix gene (M300), and H5N1 avian influenza virus (HA300)-was successfully expressed by the two-plasmid coexpression system and was demonstrated to have all of the characteristics of armored RNA. We evaluated the 3V armored L-RNA as a calibrator for multiple virus assays. We used the WHO International Standard for HCV RNA (NIBSC 96/790) to calibrate the chimeric armored L-RNA, which was diluted by 10-fold serial dilutions to obtain samples containing 10(6) to 10(2) copies. In conclusion, the approach we used for armored L-RNA preparation is practical and could reduce the labor and cost of quality control in multiplex RNA virus assays. Furthermore, we can assign the chimeric armored RNA with an international unit for quantitative detection.

Characterization of the gene delivery properties of baculoviral-based virosomal vectors.

The current study reports the production of baculoviral-virosomal vectors consisting of lipoplexes and of the viral glycoprotein (GP64) of baculovirus Autographa californica multiple nucleopolyhdrovirus (AcMNPV). This study demonstrates that such complexes have an increased transfection capability in a number of cells, including undifferentiated H9 human embryonic stem H9hES cells compared to lipoplexes alone. The GP64-mediated enhancement of gene transfer of lipoplexes is inhibited by the addition of anti-GP64 neutralizing antibody and by a modified GP64 protein, but is however less potent than vesicular stomatitis virus glycoprotein (VSV-G)-mediated enhancement of gene transfer of lipoplexes. This difference may be explained in part by the dissimilarity in the fusogenic properties of their respective viral glycoprotein.

Optimisation of secretion of recombinant HBsAg virus-like particles: Impact on the development of HIV-1/HBV bivalent vaccines.

The hepatitis B surface antigen (HBsAg) assembles into virus-like particles (VLPs) that can be used as carrier of immunogenic peptides for the development of bivalent vaccine candidates. It is shown here that by respecting certain qualitative features of mammalian preS1 and preS2 protein domains upstream of HBsAg, foreign sequences can be inserted in their place while maintaining efficient secretion of VLPs. A polyepitope bearing HIV-1 epitopes restricted to the HLA-A*0201 class I allele was optimised for secretion as an HBsAg fusion protein by counterbalancing the generally hydrophobic class I epitopes with hydrophilic spacers, eliminating epitopes bearing cysteine residues, limiting the number of internal methionine residues to a minimum and adopting Homo sapiens codon usage. The optimised HIV-1 polyepitope-HBsAg recombinant protein with up to 138 residues assembled into efficiently secreted recombinant VLPs. DNA immunisation in HLA-A*0201 and HLA-A*0201/HLA-DR1 transgenic mice resulted in the recovery of humoral response against the carrier and enhanced levels of HIV-1 specific CD8(+) T lymphocyte activation. Efficient self-assembly of recombinant HBsAg VLPs opens up the possibility of making efficient bivalent HBV/HIV vaccine candidates, which is particularly apposite given that the two viruses are frequently associated.

Virus-like particles production in green plants.

Viruses-like particles (VLPs), assembled from capsid structural subunits of several different viruses, have found a number of biomedical applications such as vaccines and novel delivery systems for nucleic acids and small molecules. Production of recombinant proteins in different plant systems has been intensely investigated and improved upon in the last two decades. Plant-derived antibodies, vaccines, and microbicides have received great attention and shown immense promise. In the case of mucosal vaccines, orally delivered plant-produced VLPs require minimal processing of the plant tissue, thus offering an inexpensive and safe alternative to more conventional live attenuated and killed virus vaccines. For other applications which require higher level of purification, recent progress in expression levels using plant viral vectors have shown that plants can compete with traditional fermentation systems. In this review, the different methods used in the production of VLPs in green plants are described. Specific examples of expression, assembly, and immunogenicity of several plant-derived VLPs are presented.

Cervical cancer-causing human papillomaviruses have an alternative initiation site for the L1 protein.

All known sequences of the DNA encoding the major cervical cancer-causing human papillomavirus type 16 (HPV16) L1 capsid protein contain initiation codons which would allow translation to begin at either nucleotide 5559 or 5637. However the formation of virus-like particles (VLPs) only occurs efficiently when the initiation codon at nucleotide 5637 is used for in vitro expression studies. This knowledge, in concert with the fact that virions have not been observed in HPV16-infected epithelium, raises the notion that the major L1 translation product in this HPV type may be largely confined to initiation at nucleotide 5559. Sequence analysis of various HPV types associated with particular clinical outcomes has revealed that L1 sequences of the major cervical cancer-associated viruses generally possess the ability to encode a longer translation product whilst the non-cancer-causing viruses do not. Equally intriguing, the upstream initiation codon is always separated by 78 nucleotides from the initiation codon that produces L1 protein which efficiently assembles into VLPs. We speculate that the longer L1 protein could play a role in the development of cervical carcinoma and that HPVs with the potential to cause cervical cancer may be identified by the presence of an in-frame ATG situated 78 nucleotides upstream.