Abstract
The VLPNPV 2014 Conference that was convened at the Salk institute was the second conference of its kind to focus on advances in production, purification, and delivery of virus-like particles (VLPs) and nanoparticles. Many exciting developments were reported and discussed in this interdisciplinary arena, but here we report specifically on the contributions of plant-based platforms to VLP vaccine technology as reported in the section of the conference devoted to the topic as well in additional presentations throughout the meeting. The increasing popularity of plant production platforms is due to their lower cost, scalability, and lack of contaminating animal pathogens seen with other systems. Reports include production of complex VLPs consisting of 4 proteins expressed at finely-tuned expression levels, a prime-boost strategy for HIV vaccination using plant-made VLPs and a live viral vector, and the characterization and development of plant viral nanoparticles for use in cancer vaccines, drug delivery, and bioimaging.
Abbreviations
| Ab | = | antibody |
| BPV | = | bovine papillomavirus |
| BTV | = | Bluetongue virus |
| CPMV | = | cowpea mosaic virus |
| ELISA | = | enzyme-linked immunosorbent assay |
| HBV | = | Hepatitis B virus |
| HER2 | = | human epidermal growth factor receptor 2 (also called c-ErbB-2) |
| HIV | = | human immunodeficiency virus |
| HT | = | HyperTrans |
| Ig | = | immunoglobulin |
| i.p. | = | intraperitoneal |
| NPV | = | nano-particle vaccine |
| MPR | = | membrane proximal region |
| PEG | = | polyethylene glycol |
| PVX | = | potato virus X |
| SNP | = | spherical nanoparticle |
| TMV | = | tobacco mosaic virus |
| UTR | = | untranslated region |
| VLP | = | virus-like particle |
| VNP | = | viral nanoparticle |
Introduction
The second meeting of the Virus-like Particle and Nano-particle Vaccines International Conference brought together dozens of renowned experts from across the world to the Salk Institute for Biological Sciences in La Jolla, CA. Vaccines based on virus-like particles (VLPs) are promising safe alternatives to vaccines based on live attenuated or inactivated pathogens but they often require administration of high doses and multiple boosts for optimum immunogenicity. Our understanding of the immunological mechanisms that underlie the promise of VLP vaccines is incomplete and suggest there is much room for improvement. Exciting reports presented in the meeting, which described in-depth analyses of the immunological consequences of VLP administration, not only start to fill-in some of these knowledge gaps, but also bear important practical implications that can be quickly applied and tested. For example, mucosal delivery of VLPs is more effective in priming immune responses than their systemic administration.Citation1,2 Such advances aside, it is clear that for VLPs to make a significant impact on large-scale vaccine programs, cost-effective expression systems that can provide high yield without compromising quality is required. Therefore, much presentation and discussion time was devoted at VLPNPV meeting for innovation in producing, purifying, and characterizing VLPs and NPVs.
The use of plants as protein expression platforms is a relatively new but rapidly growing field with a variety of established systems for plant-based production of biologics such as antibodies (Abs), enzymes and vaccine antigens including VLPs.Citation3,4 In fact, the use of the technology received much public and media attention very recently when a plant-produced cocktail of monoclonal Abs were effective in recovery of 2 Ebola patients.Citation5-7 Plants offer advantages over other production platforms, such as mammalian and insect cells, due to their scalability while maintaining a lower cost than that of cell culture and avoiding possible contamination with mammalian pathogens. Here we report advances in plant-based VLP production technology. Recently developed deconstructed viral based expression vectors derived from ssDNA and ssRNA plant viruses improved both speed of production and yield.Citation8-12 These vectors have permitted the expression of increasingly complex VLPs requiring simultaneous assembly of multiple virion protein subunits present at specific ratios.Citation13 Furthermore, plant viral nanoparticles can serve as carrier particles to present specific vaccination epitopes or for applications in nanomedicine.Citation14,15 Many vaccination trials have been conducted showing the immunogenicity of plant-made VLPs for many important human and animal diseases including hepatitis B virus, human papillomavirus, HIV-1, and foot-and-mouth disease virus (for recent reviews see refs. 1–2). Conference attendants received excellent thorough updates on 2 plant-produced VLPs that are among the most advanced in the development pipeline – VLPs of noroviruses Citation16 and influenza viruses.Citation17 These excellent lectures were given, respectively, by Melissa Herbst-Kralovetz (University of Arizona Medical School, Phoenix, AZ, USA) and Marc-André D’Aoust (Medicago Inc., Bureau, QC, Canada) in more general sessions during the conference and will not be discussed further, in this report, that will cover updates and examples of each of plant-based technologies presented at Session 5 dedicated to the topic.
Plant-production of complex VLPs: the case of bluetongue virus
Bluetongue virus (BTV) is an insect-borne dsRNA virus of the Reoviridae causing potentially fatal disease in wild and domesticated ruminants in temperate climates.Citation18 Previously restricted to sub-Saharan Africa, several significant outbreaks in Eurasia (e.g. the 2006–2007 outbreak spreading to France, Germany, Belgium, and Luxembourg infecting over 2000 herds with almost 50% mortality of the infected sheep),Citation19,20 raise global concerns. While attenuated and inactivated whole virus vaccines currently exist, common and serious side effects often occur which leads to questions of safety when vaccinating on a large scale and must be given as a cocktail to protect against several of the 26 known serotypes.Citation21-24 George Lomonossoff (John Innes Center, Colney, Norwich, UK) presented advances his group has made in the production of complex BTV VLPs for vaccination.
Using their novel cowpea mosaic virus (CPMV)-based vectors, the Lomonossoff group previously reported on successful production of simple VLPs consisting of a single capsid protein in plants. For example, they were able to demonstrate expression and assembly of bovine papillomavirus (BPV) VLPs that consist of the viral L1 protein and of the Hepatitis B virus (HBV) core particle consisting of the HBV core antigen.Citation25,26 However, BTV has a more complex virion structure than either BPV or HBV involving 4 different proteins at specific ratios arranged in concentric spheres. Viral proteins VP3 and VP7 form a non-immunogenic inner shell with approximately 120 and 780 copies, respectively. The outer shell that is absolutely required for immunogenicity, consists of VP5 and VP2 with 360 and 180 copies, respectively.Citation27-29
In his talk Lomonossof described the recently publishedCitation13 plant-based production system, for which vectors have been specifically designed to control the ratios of each of the 4 proteins for BTV-8. In an early stage of the research, genes encoding the 4 proteins were cloned into individual CPMV pEAQ-HyperTrans (HT) expression vectors.Citation11,12,26 The pEAQ vectors, co-expressing the P19 anti-silencing gene, are unique in that while non-replicating can still provide rapid, high-level protein expression in plants with the possibility of delivering multiple vectors per cell. The HT mutation in the vectors removes an ATG in the 5’UTR thus increasing expression, likely by reducing leaky scanning upstream of the gene start codon.Citation11,12 Introduction of the BTV structural genes using 4 individual vectors resulted in high level of co-expression of the proteins, which assembled into a myriad of incomplete VLPs as visualized by electron microscopy.Citation13 Specifically, they observed an overabundance of non-immunogenic VP3/VP7 core-like particles and VP3 subcore-like particles, The simple, but elegant solution to this problem was solved by down-regulating expression of VP3 by re-introducing the 5′UTR ATG in the pEAQ vector, thus eliminating the HyperTrans enhancement. This shifted assembly of VLPs in favor of mature VLPs at the expense of the partially-assembled or incomplete particles, allowing a more homogeneous preparation of the immunogenic mature VLPs.Citation13 Importantly, these VLPs were tested in sheep and were able to elicit Ab titers comparable to the current vaccine and protect the vaccinated animals against a challenge, thus providing a safer, viable vaccine candidate.Citation13 Lomonossoff gave a compelliing demonstration of a powerful new tool to fine-tune expression levels of each individual protein subunits required at a prescribed stoichiometry and shift the accumulation toward the desired assembly product (e.g., immunogenic VLPs).
VLPs as a display platform
The CPMV based pEAQ-HT expression system also featured in the talk of Hadrien Peyret (John Innes Center, Colney, Norwich, UK). Peyret used this technology to create HBV core VLPs displaying the variable region of a camelid Ab. The Ab fragment is genetically engineered into the outward-facing e1 immunogenic loop of the HBV capsid protein. The resulting VLPs, that Peyret affectionately calls ‘tandibodies’, can then be mixed with the cognate antigen to rapidly create an antigen display platform for a given protein. The beauty of tandibodies is that they potentially should overcome the limitation in the size of antigens displayed on HBV core particles. A much larger protein can be tethered to the particle by binding to the Ab fragment than the relatively short peptides that can typically be inserted into the capsid protein without negatively impacting VLP assembly. Peyret presented visually compelling evidence to the ability of anti-GFP tandibodies to display GFP. Attempts to repeat the success with a medically-relevant protein (HIV-1 envelope protein's surface subunit gp120) are still tenuous. The reasons for this are unclear, but poor accessibility of the Ab fragments on the surface of the tandibody to the gp120 cargo is a reasonable assumption. This protein is much larger than GFP and tends to form stable trimers, increasing the size of this cargo protein even further potentially masking the epitope. Research is ongoing to demonstrate the feasibility of the system beyond the GFP proof of principle. The potential value of this clever system to rapidly engineer an established VLP capsid with a known Ab fragment to create a full-protein display system is enormous. If used for vaccination, this could increase the breadth of an immune response by presenting multiple epitopes to the immune system instead of the limited epitopes displayed in peptide vaccines.
Vaccination with HIV-1 Gag/dgp41 VLPs
HIV-1 represents one of the most elusive viruses for established vaccination strategies to-date and, despite the onset of antiretroviral therapies, continues to spread each year with the largest incidence rates primarily in the developing world. The most successful HIV vaccine trial (RV144) used a non-replicating canarypox prime followed by a mixed virus plus soluble protein boost and achieved a modest 31% efficacy with few identified correlates of protection.Citation30-33 In our talk and poster presentation, (TSM and LRM, Arizona State University, Tempe, Arizona, USA) we reported a vaccination strategy set to improve upon the results of RV144 using plant-based Gag/dgp41 VLPs and a replicating viral vector. HIV infected individuals with T cell responses to Gag show increased control of viral replication.Citation34 Additionally, there is great interest in inducing rare, but naturally occuring broadly neutralizing Abs, such as those targeting the membrane proximal region (MPR) of gp41 including 2F5, 4E10, and 10E8.Citation35-37
Previous work in our lab has shown that the MPR of gp41 is immunogenic in mice and can elicit transcytosis blocking Abs both when attached to the cholera toxin B subunit and when expressed in plants in the context of a Gag VLP.Citation38-40 For VLPs, a deconstructed version of gp41 (dgp41) is used which truncates the N-terminus to the membrane proximal region to better expose this neutralizing Ab target. Gag/dgp41 VLPs are produced using stable transgenic Gag plants with transient dgp41 expression using the MagnICON TMV-based expression vectors.Citation38 VLPs present an advantage over soluble protein used in RV144 by presenting the antigen in the native context of an enveloped Gag matrix, and the membrane context is known to be required for eliciting 2F5 broadly neutralizing Abs.Citation41,42 While VLPs alone are capable of inducing serum IgG and mucosal IgA to Gag and dgp41, a replicating viral vector was added in an effort to enhance T cell responses.
NYVAC is a highly attenuated vaccinia virus with 18 specific open reading frame deletions hindering replication in human cells.Citation43 NYVAC-KC is a further modification which re-introduced 2 genes, K1L and C7L, to restore the ability to replicate in human cells and enhance immunogenicity while maintaining attenuation.Citation44,45 These viruses were engineered to express Gag and dgp41 in separate constructs. Replication competent viral vectors, including many vaccine strains of vaccinia,Citation46 are of great interest for use as vaccination strategies to enhance T cell responses. Recently, a persistent CMV vector expressing HIV-1 envelope protein was capable of protecting non-human primates from challenge with SIV, demonstrating the potential of replication competent viral vectors for an HIV vaccination strategy.Citation47,48
The Mor group tested this heterologous prime-boost strategy through combining plant-produced Gag/dgp41 VLPs with the replicating vaccinia virus vectors. C57BL/6 mice were immunized with a single virus prime followed by 2 boosts spaced 45 d apart of either VLP or virus plus VLP. Serum IgG and fecal and vaginal IgA Abs were detected using ELISA to either Gag p24 or gp41 MPR peptide. The study showed that animals primed with vaccinia virus did not show strong Ab responses to Gag or dgp41 until after a VLP boost. Mucosal IgA responses were weak but significant for fecal IgA specific for dgp41 MPR. IgA responses could possibly be improved using a mucosal route of immunization instead of i.p. as administered here. Furthermore, final CD8 T cell responses in the spleen showed a significant increase in Gag-specific responses in groups primed with virus but boosted with both virus and VLPs. Endpoint titers for serum IgG against vaccinia virions showed higher titers of vaccinia-specific Abs after 3 doses of virus presenting the possibility of neutralization of the viral vector if multiple boosts are required. While this represents a unique combination of strategies for HIV vaccination, further work is necessary to optimize this vaccination regimen and possibly choose a vaccinia vector which is mouse-adapted to enhance immunogenicity over NYVAC-KC, a strain engineered for replication in humans.
Plant viral nanoparticles: targeting through design
Plant viruses are natural carriers of cargo with built-in, often cell type-specific delivery mechanisms, and are therefore attracting more and more attention for their potential untility in the growing field of nanomedicine for rapid and targeted delivery of drugs and contrast agents for imaging.Citation14,15,49 These viral nanoparticles (VNPs) are considered safe for use in humans due to their lack of pathogenicity in animals and typically faster clearance rates than synthetic nanoparticles.Citation15,50,51 Each virus has unique properties which determine biodistribution, circulation half-life, tumor homing, and interactions with immune cells. These characteristics are influenced by both size and shape, aspect ratio, surface chemistry, and electrokinetic (ζ, zeta) potential.Citation52-55 Once thoroughly characterized in vitro and in vivo, this knowledge will contribute to the design of VNPs for specific applications in drug delivery, imaging, and vaccination.
Nicole Steinmetz (Case Western Reserve University, Cleveland, Ohio, USA) discussed her lab's recent findings and strategies in the design of a cancer vaccine targeting the human epidermal growth factor receptor 2 (HER2, also known as c-Erbb-2) using potato virus X nanoparticles as an antigen display platform. HER2 is a proto-oncogene and a predominant tumor associated antigen found to be overexpressed in various types of cancer cells.Citation56 HER2 has been extensively studied in breast cancer patients with multiple treatment and vaccination strategies ranging from monoclonal Ab administration and peptide vaccines to whole tumor and dendritic cell based vaccines.Citation57
Potato virus X (PVX) provides a novel and appealing platform choice due to its specific VNP properties and is currently being developed and analyzed by this group.Citation14 PVX is a high aspect ratio filament (515 × 13nm) with a positive ζ potential.Citation58 Experiments with many avian embryo and mouse xenograft human cancer models show increased accumulation and tumor penetration by PVX when compared with low aspect ratio spherical CPMV particles.Citation58 The distinct biodistribution profiles of the 2 VNPs shows PVX has increased presence in the spleen while CPMV is primarily in the liver.Citation58 These differences show an interesting influence of shape and surface charge on bioavailability, which would determine the specific applications for which they might be used. Based on these data, they chose the PVX platform to display antigens for a HER2 cancer vaccine because it preferentially targets the spleen for enhanced immunogenicity over spherical CPMV particles.
Interestingly, other studies discussed at the meeting, and recently published, show that increasing the aspect ratio of CPMV spheres through dimerization with polyethylene glycol (PEG) maleimide linkers enhanced particle uptake by >2.5-fold in HeLa cells.Citation59 Thus, it is possible to artificially increase aspect ratio to alter cellular interactions with VNPs for specific applications. George Lomonossoff (John Innes Center, Colney, Norwich, UK) showed cryo-electron microscopy images from similar, but empty, CPMV particles lacking RNA as a non-replicating system in which the coat protein can be chemically or genetically modified. These particles are produced in plants and can be subjected to mineralization.Citation60-62 They are currently developing a technique for genetically modifying the empty VLPs to display the integrin-binding RGD peptide for potential use in targeting for drug delivery or imaging applications.
The ability to manipulate plant VLPs in vitro was also explored by Steinmetz. In her talk, she described the direct manipulation of shape for tobacco mosaic virus (TMV) VNPs.Citation63 TMV rod VNPs of high aspect ratio (300 × 18 nm) were thermally denatured to create RNA-free spherical nanoparticles (SNPs) with a lower aspect ratio ranging from 50–800 nm where final size was dependent upon starting protein concentration.Citation63 Additional data shown at the conference described a technique to manipulate length of TMV rods by altering the size of the RNA with which it interacts recalling old experiments (see the review by ButlerCitation64 and references therein). This strategy represents a more precise method for controlling size and altering aspect ratio along a more continuous gradient. Both rod VNPs and SNPs have similar negative zeta potentials due to their construction from the same coat protein, thus providing a unique platform in which the effect of size and shape on nanoparticle properties can be directly assessed without confounding variables. Biological systems analysis revealed TMV rods have a 4-fold increase of particles remaining in circulation 60 min post injection.Citation63 Furthermore, both rods and SNPs co-localize with macrophages in the marginal zone of the spleen after 4 hours but primarily rods are found co-localizing with B cell markers after 24 hours.Citation63 This holds implications for choice of clinical applications: rods may be more effective at immunological interactions by evading rapid macrophage clearance experienced by SNPs, which represents a more advantageous property for imaging techniques. These data also support the choice of the high aspect ratio PVX filamentous particles for a HER2 cancer vaccine because it appears their increased circulation time and interaction with immunologically relevant cells in the spleen may contribute to an immune response. Collectively, these studies demonstrate novel technologies for directly manipulating VNP size and shape to direct a specific change in characteristics and provide crucial information for choosing the right VNP for a particular clinical application.
Summary
During VLPNPV 2014, more than in the inaugural meeting that took place 2 y ago in Cannes, France, plant-based technologies were extensively demonstrated to be at the forefront of innovation in the emerging field of VLPs and NPV. Plants find utility as a production platform for VLPs of mammalian pathogens and vaccination strategies involved using such VLPs are created. There's also a growing interest in taking advantage of plant virus VLPs as nanoparticles for multiple uses in diagnosing and treating major human and animal diseases. The meeting provided an excellent opportunity for thoughtful discussion, creative brainstorming and facilitating old and new collaborations in this rapidly advancing field.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
4–6 June 2014 The Salk Institute for Biological Sciences, La Jolla, CA USA.
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