Stability Of Virus-like Particles Research Articles

Stability and integrity of self-assembled bovine parvovirus virus‑like particles (BPV‑VLPs) of VP2 and combination of VP1VP2 assisted by baculovirus-insect cell expression: a potential logistical platform for vaccine deployment

BackgroundBovine parvovirus (BPV) is an autonomous DNA virus with a smaller molecular size and subtle differences in its structural proteins, unlike other animal parvoviruses. More importantly, this virus has the potential to produce visible to silent economic catastrophes in the livestock business, despite receiving very little attention. Parvoviral virus-like particles (VLPs) as vaccines and as logistical platforms for vaccine deployment are well studied. However, no single experimental report on the role of VP1 in the assembly and stability of BPV-VLPs is available. Furthermore, the self-assembly, integrity and stability of the VLPs of recombinant BPV VP2 in comparison to VP1 VP2 Cap proteins using any expression method has not been studied previously. In this study, we experimentally evaluated the self-assembling ability with which BPV virus-like particles (VLPs) could be synthesized from a single structural protein (VP2) and by integrating both VP2 and VP1 amino acid sequences.MethodsIn silico and experimental cloning methods were carried out. His-tagged and without-His-tag VP2 and V1VP2-encoding amino acid sequences were cloned and inserted into pFastbacdual, and insect cell-generated recombinant protein was evaluated by SDS‒PAGE and western blot. Period of infectivity and expression level were determined by IFA. The integrity and stability of the BPV VLPs were evaluated by transmission electron microscopy. The secondary structure of the BPV VLPs from both VP2 and V1VP2 was analyzed by circular dichroism.ResultsOur findings show that VP2 alone was equally expressed and purified into detectable proteins, and the stability at different temperatures and pH values was not appreciably different between the two kinds of VLPs. Furthermore, BPV-VP2 VLPs were praised for their greater purity and integrity than BPV-VP1VP2 VLPs, as indicated by SDS‒PAGE. Therefore, our research demonstrates that the function of VP1 has no bearing on the stability or integrity of BPV-VLPs.ConclusionsIn summary, incredible physiochemically stable BPV VP2-derived VLPs have been found to be promising candidates for the development of multivalent vaccines and immunodiagnostic kits against enteric viruses and to carry heterogeneous epitopes for various economically important livestock diseases.

Virus-like Particles Armored by an Endoskeleton.

Many virus-like particles (VLPs) have good chemical, thermal, and mechanical stabilities compared to those of other biologics. However, their stability needs to be improved for the commercialization and use in translation of VLP-based materials. We developed an endoskeleton-armored strategy for enhancing VLP stability. Specifically, the VLPs of physalis mottle virus (PhMV) and Qβ were used to demonstrate this concept. We built an internal polymer "backbone" using a maleimide-PEG15-maleimide cross-linker to covalently interlink viral coat proteins inside the capsid cavity, while the native VLPs are held together by only noncovalent bonding between subunits. Endoskeleton-armored VLPs exhibited significantly improved thermal stability (95 °C for 15 min), increased resistance to denaturants (i.e., surfactants, pHs, chemical denaturants, and organic solvents), and enhanced mechanical performance. Single-molecule force spectroscopy demonstrated a 6-fold increase in rupture distance and a 1.9-fold increase in rupture force of endoskeleton-armored PhMV. Overall, this endoskeleton-armored strategy provides more opportunities for the development and applications of materials.

Surface Cross-Linking by Macromolecular Tethers Enhances Virus-like Particles' Resilience to Mucosal Stress Factors.

Virus-like particles (VLPs) are emerging as nanoscaffolds in a variety of biomedical applications including delivery of vaccine antigens and cargo such as mRNA to mucosal surfaces. These soft, colloidal, and proteinaceous structures (capsids) are nevertheless susceptible to mucosal environmental stress factors. We cross-linked multiple capsid surface amino acid residues using homobifunctional polyethylene glycol tethers to improve the persistence and survival of the capsid to model mucosal stressors. Surface cross-linking enhanced the stability of VLPs assembled from Acinetobacter phage AP205 coat proteins in low pH (down to pH 4.0) and high protease concentration conditions (namely, in pig and mouse gastric fluids). Additionally, it increased the stiffness of VLPs under local mechanical indentation applied using an atomic force microscopy cantilever tip. Small angle X-ray scattering revealed an increase in capsid diameter after cross-linking and an increase in capsid shell thickness with the length of the PEG cross-linkers. Moreover, surface cross-linking had no effect on the VLPs' mucus translocation and accumulation on the epithelium of in vitro 3D human nasal epithelial tissues with mucociliary clearance. Finally, it did not compromise VLPs' function as vaccines in mouse subcutaneous vaccination models. Compared to PEGylation without cross-linking, the stiffness of surface cross-linked VLPs were higher for the same length of the PEG molecule, and also the lifetimes of surface cross-linked VLPs were longer in the gastric fluids. Surface cross-linking using macromolecular tethers, but not simple conjugation of these molecules, thus offers a viable means to enhance the resilience and survival of VLPs for mucosal applications.

An integrative and multiscale approach to study dengue virus-like particle dynamics.

Dengue virus is an enveloped virus which infects millions of people worldwide. The only available vaccine has low efficacy and is recommended only for individuals with prior dengue infection. Virus-like particles (VLPs) are promising platforms as vaccine candidates due to their non-infectious and highly immunogenic nature. These particles lack a genomic core and are highly unstable and heterogeneous in size. This motivates the characterization of the biophysical and structural properties of VLPs to facilitate the rational production of stable and homogenous particles. Dengue VLPs elicits neutralizing antibodies similar to infectious virions. Dengue VLPs are enveloped particles in which sixty envelope (E) and membrane (M) proteins are embedded in a lipid vesicle. Similarly, to the dengue virus, the VLPs undergo a maturation process, involving large conformational changes, leading to exposure of epitopes that subsequently determine the immune response. In this work, the structure and intrinsic dynamics of VLPs and their conformational transitions were investigated using integrative modeling and molecular simulations. To gain atomistic insights into the maturation process and to facilitate the development of stable enveloped VLPs, we employed an integrative approach combining multiscale modeling and simulations with experimental data from cryo-electron microscopy, lipidomics, and secretion assays. Coarse-grained simulations of mature and immature VLPs revealed the effective number of lipids that result in optimally stable and spherical particles. Furthermore, these models were used as a platform to simulate the transition between conformational states in order to understand the maturation process in unprecedented detail. Finally, atomistic MD simulations performed on native dengue and dengue-Japanese encephalitis virus chimeric E/M proteins and their mutants highlighted the role of protein-lipid interactions in governing VLP stability. This integrative approach aims towards the future development of stable and immunogenic next-generation vaccines.

Improvement of Modular Protein Display Efficiency in SpyTag-Implemented Norovirus-like Particles.

Genetic fusion and chemical conjugation are the most common approaches for displaying a foreign protein on the surface of virus-like particles (VLPs); however, these methods may negatively affect the formation and stability of VLPs. Here, we aimed to develop a modular display platform for protein decoration on norovirus-like particles (NoV-LPs) by combining the NoV-LP scaffold with the SpyTag/SpyCatcher bioconjugation system, as the NoV-LP is an attractive protein nanoparticle to carry foreign proteins for various applications. The SpyTagged-NoV-LPs were prepared by introducing SpyTag peptide into the C-terminus of the norovirus VP1 protein. To increase surface exposure of the SpyTag peptide on the NoV-LPs, two or three repeated extension linkers (EAAAK) were inserted between the SpyTag peptide and VP1 protein. Fluorescence proteins, EGFP and mCherry, were fused to SpyCatcher and employed as SpyTag conjugation partners. These VP1-SpyTag variants and SpyCatcher-fused EGFP and mCherry were separately expressed in silkworm fat bodies and purified. This study reveals that adding an extension linker did not disrupt the VLP formation; instead, it increased the particle size by 4-6 nm. The conjugation efficiency of the VP1-SpyTag variants with the extended linker improved from ∼15-35 to ∼50-63% based on the densitometric analysis, while it was up to 77% based on an optical quantification of EGFP and mCherry. Results indicate that the linker causes the SpyTag peptides to be positioned further away from the C-termini of VP1 and potentially increases the exposure of the SpyTag to the outer surface of the NoV-LPs, allowing more SpyTag/SpyCatcher complex formation on the VLP surface. Our study provides a strategy for enhancing the conjugation efficiency of NoV-LP and demonstrates the platform's utility for developing vaccines or functional nanoparticles.

Production of cytoplasmic type citrus leprosis virus-like particles by plant molecular farming

Many plant virus-like particles (VLPs) utilized in nanotechnology are 30-nm icosahedrons. To expand the VLP platforms, we produced VLPs of Cytoplasmic type citrus leprosis virus (CiLV-C) in Nicotiana benthamiana. We were interested in CiLV-C because of its unique bacilliform shape (60–70 nm × 110–120 nm). The CiLV-C capsid protein (p29) gene was transferred to the pTRBO expression vector transiently expressed in leaves. Stable VLPs were formed, as confirmed by agarose gel electrophoresis, transmission electron microscopy and size exclusion chromatography. Interestingly, the morphology of the VLPs (15.8 ± 1.3 nm icosahedral particles) differed from that of the native bacilliform particles indicating that the assembly of native virions is influenced by other viral proteins and/or the packaged viral genome. The smaller CiLV-C VLPs will also be useful for structure–function studies to compare with the 30-nm icosahedrons of other VLPs.

Flower-like mesoporous silica nanoparticles as an antigen delivery platform to promote systemic immune response

Virus-like particles (VLPs), a kind of superior subunit vaccine, are assembled from the viral structural proteins with similar capsids to viruses. However, the efficiency of cell uptake is not satisfactory. We prepared flower-like mesoporous silica nanoparticles (SiNPs) with large pore channels and interior cavities to solve the problem. The highly loaded VLPs-SiNPs composites not only enhanced the stability of VLPs, but also delivered antigen to cells and improved the cellular uptake efficiency. Compared with naked VLPs, mice intramuscularly immunized with the VLPs-SiNPs composite induced higher specific antibodies, greater lymphocyte activation and higher level of cytokine secretion. Moreover, the VLPs-SiNPs composite as vaccine also promoted mucosal immune response through intranasal immune pathway. Therefore, the VLPs-SiNPs enable to induce strong cellular, humoral, and slight mucosal immune response through different immunization routes. These results are potentially useful for vaccine formulations and may provide further reference for vaccine design and delivery systems.

Four Simple Biomimetic Mineralization Methods to Improve the Thermostability and Immunogenicity of Virus-like Particles as a Vaccine against Foot-and-Mouth Disease

The need for a cold chain system during storage and transport substantially increases the cost of vaccines. Virus-like particles (VLPs) are among the best countermeasures against foot and mouth disease virus (FMDV). However, VLPs are composed of pure proteins, and thus, are susceptible to heat. To address this problem, four simple biomimetic mineralization methods with the use of calcium phosphate were developed to improve heat tolerance via biomineralization. The results showed that biomineralization can significantly improve the heat resistance of VLPs. The biomineralized VLPs can be stored at low as 25 °C for eight days, and 37 °C for four days. Animal experiments showed that biomineralization had no effect on the immunogenicity of VLPs or the expression of specific antibodies (Abs) and neutralizing Abs. Even after heat treatment at 37 °C for four days, the biomineralized VLPs remained immunogenic and produced highly specific and neutralizing Abs with a high rate of protection. These results suggest that these biomineralization approaches can promote the thermal stability of VLPs against and significantly reduce dependence on cold storage and delivery systems.

Effect of amino acid site modification on stability of foot-and-mouth disease virus-like particles

The stability of virus-like particles (VLPs) is currently the main factor affecting the quality of foot-and-mouth disease VLPs vaccines. In order to further improve the quality of the VLPs vaccine of foot-and-mouth disease (FMD), three amino acid modification sites were designed and screened through kinetic analysis software, based on the three-dimensional structure of FMDV. The three mutant recombinant plasmids were successfully prepared by the point mutation kit, transformed into Escherichia coli strain BL21 and expressed in vitro. After purification by Ni ion chromatography column, SDS-PAGE proved that the three amino acid mutations did not affect the expression of the target protein. The results of the stability study of three FMD mutant VLPs obtained by in vitro assembly show that the introduction of internal hydrophobic side chain amino acids made the morphology of VLPs more uniform (N4017W), and their stability was significantly improved compared to the other two VLPs. The internal hydrophobic force of the capsid contributes to the formation of VLPs and helps to maintain the stability of the capsid, providing new experimental ideas for improving the quality of VLPs vaccines, and helping to promote the development of VLPs vaccines.

Virus-Like Particles Produced Using the Brome Mosaic Virus Recombinant Capsid Protein Expressed in a Bacterial System.

Virus-like particles (VLPs), due to their nanoscale dimensions, presence of interior cavities, self-organization abilities and responsiveness to environmental changes, are of interest in the field of nanotechnology. Nevertheless, comprehensive knowledge of VLP self-assembly principles is incomplete. VLP formation is governed by two types of interactions: protein–cargo and protein–protein. These interactions can be modulated by the physicochemical properties of the surroundings. Here, we used brome mosaic virus (BMV) capsid protein produced in an E. coli expression system to study the impact of ionic strength, pH and encapsulated cargo on the assembly of VLPs and their features. We showed that empty VLP assembly strongly depends on pH whereas ionic strength of the buffer plays secondary but significant role. Comparison of VLPs containing tRNA and polystyrene sulfonic acid (PSS) revealed that the structured tRNA profoundly increases VLPs stability. We also designed and produced mutated BMV capsid proteins that formed VLPs showing altered diameters and stability compared to VLPs composed of unmodified proteins. We also observed that VLPs containing unstructured polyelectrolyte (PSS) adopt compact but not necessarily more stable structures. Thus, our methodology of VLP production allows for obtaining different VLP variants and their adjustment to the incorporated cargo.

Antigenicity study of the yeast-generated human parvovirus 4 (PARV4) virus-like particles

Human parvovirus 4 (PARV4) is a novel tetraparvovirus that was isolated from intravenous drug users in 2005. Recombinant PARV4 capsid protein VP2 can form stable virus-like particles (VLPs) in yeast. These VLPs could act as antigen carriers during vaccine development. Therefore, the information about PARV4 VP2 VLP antigenic sites could advance further research in this area. In this work, human parvovirus 4 VLPs obtained from yeast were used to generate monoclonal antibodies (mAbs) in mice. Epitope mapping of the obtained mAbs showed at least three distinct antigenic sites of the VP2 protein. On top of that, molecular cloning was used to replace PARV4 VP2 antigenic sites with heterologous peptides. The chimeric PARV4 VLPs bearing polyhistidine inserts obtained from yeast were observed using electron microscopy while polyhistidine-specific antibodies detected heterologous peptides of the chimeric VP2 proteins.

Capsid destabilization and epitope alterations of human papillomavirus 18 in the presence of thimerosal

Thimerosal has been widely used as a preservative in drug and vaccine products for decades. Due to the strong propensity to modify thiols in proteins, conformational changes could occur due to covalent bond formation between ethylmercury (a degradant of thimerosal) and thiols. Such a conformational change could lead to partial or even complete loss of desirable protein function. This study aims to investigate the effects of thimerosal on the capsid stability and antigenicity of recombinant human papillomavirus (HPV) 18 virus-like particles (VLPs). Dramatic destabilization of the recombinant viral capsid upon thimerosal treatment was observed. Such a negative effect on the thermal stability of VLPs preserved with thimerosal was shown to be dependent on the thimerosal concentration. Two highly neutralizing antibodies, 13H12 and 3C3, were found to be the most sensitive to thimerosal treatment. The kinetics of antigenicity loss, when monitored with 13H12 or 3C3 as probes, yielded two distinctly different sets of kinetic parameters, while the data from both monoclonal antibodies (mAbs) followed a biphasic exponential decay model. The potential effect of thimerosal on protein function, particularly for thiol-containing proteinaceous active components, needs to be comprehensively characterized during formulation development when a preservative is necessary.

Synthesis and Assembly of Hepatitis B Virus-Like Particles in a Pichia pastoris Cell-Free System.

Virus-like particles (VLPs) are supramolecular protein assemblies with the potential for unique and exciting applications in synthetic biology and medicine. Despite the attention VLPs have gained thus far, considerable limitations still persist in their production. Poorly scalable manufacturing technologies and inconsistent product architectures continue to restrict the full potential of VLPs. Cell-free protein synthesis (CFPS) offers an alternative approach to VLP production and has already proven to be successful, albeit using extracts from a limited number of organisms. Using a recently developed Pichia pastoris-based CFPS system, we have demonstrated the production of the model Hepatitis B core antigen VLP as a proof-of-concept. The VLPs produced in the CFPS system were found to have comparable characteristics to those previously produced in vivo and in vitro. Additionally, we have developed a facile and rapid synthesis, assembly and purification methodology that could be applied as a rapid prototyping platform for vaccine development or synthetic biology applications. Overall the CFPS methodology allows far greater throughput, which will expedite the screening of optimal assembly conditions for more robust and stable VLPs. This approach could therefore support the characterization of larger sample sets to improve vaccine development efficiency.

Engineered hepatitis B core virus-like particle carrier for precise and personalized Alzheimer’s disease vaccine preparation via fixed-point coupling

Heterogenous aggregates of amyloid β (Aβ) and tau protein play a key role in Alzheimer’s disease (AD) progression. As the epitope profiles of Aβ and abnormally phosphorylated tau change over the course of the disease, tailor immunotherapy to specific patient groups according to the disease stage by targeting corresponding Aβ and tau species or both of tau and Aβ might improve treatment efficacy. However, epitope peptides have low immunogenicity and peptide vaccine preparation is laborious and time-consuming. We here first constructed a platform for peptide vaccine preparation by inserting SpyCatcher into the major immunodominant region (MIR) of truncated Hepatitis B core protein (HBc)(1-149). The resulted recombinant protein HBc-SpyCatcher (HBc-S) could assemble into uniform and stable virus-like particles (VLPs), and readily bind to the SpyTag conjugated epitope peptides. Several series of peptides such as linear, cyclic and phosphorylated peptides including Aβ(1-6), Aβ(1-15), cAβ(1-7), cEP1, cEP2 from β-amyloid monomer or oligomers, T294, pTau396-404, pTau422 from tau proteins, were glued onto HBc-S VLPs, forming HBc-S-peptide (HBc-S-P) VLPs and efficiently elicited specific Th2-type immune responses. When applied to AD transgenic mice, HBc-S-pTau422 alleviated cognition deficits and neuropathology progression in Tau.P301S transgenic mice. The present study provides an easy, quick, convenient and universal platform for high-throughput peptide epitope screening and personalized peptide vaccine preparation.

Effect of natural ageing and heat treatments on GII.4 norovirus binding to Histo-Blood Group Antigens

Human noroviruses (HuNoVs) are the leading cause of viral foodborne outbreaks worldwide. To date, no available methods can be routinely used to detect infectious HuNoVs in foodstuffs. HuNoVs recognize Histo-Blood Group Antigens (HBGAs) through the binding pocket (BP) of capsid protein VP1, which promotes infection in the host cell. In this context, the suitability of human HBGA-binding assays to evaluate the BP integrity of HuNoVs was studied on GII.4 virus-like particles (VLPs) and GII.4 HuNoVs during natural ageing at 20 °C and heat treatments. Our results demonstrate that this approach may reduce the over-estimation of potential infectious HuNoVs resulting from solely using the genome detection, even though some limitations have been identified. The specificity of HBGA-binding to the BP is clearly dependent on the HGBA type (as previously evidenced) and the ionic strength of the media without disturbing such interactions. This study also provides new arguments regarding the ability of VLPs to mimic HuNoV behavior during inactivation treatments. The BP stability of VLPs was at least 4.3 fold lower than that of HuNoVs at 20 °C, whereas capsids of both particles were disrupted at 72 °C. Thus, VLPs are relevant surrogates of HuNoVs for inactivation treatments inducing significant changes in the capsid structure.

Role of metallic core for the stability of virus-like particles in strongly coupled electrostatics

Electrostatic interactions play important roles in the formation and stability of viruses and virus-like particles (VLPs) through processes that often involve added, or naturally occurring, multivalent ions. Here, we investigate the electrostatic or osmotic pressure acting on the proteinaceous shell of a generic model of VLPs, comprising a charged outer shell and a metallic nanoparticle core, coated by a charged layer and bathed in an aqueous electrolyte solution. Motivated by the recent studies accentuating the role of multivalent ions for the stability of VLPs, we focus on the effects of multivalent cations and anions in an otherwise monovalent ionic solution. We perform extensive Monte-Carlo simulations based on appropriate Coulombic interactions that consistently take into account the effects of salt screening, the dielectric polarization of the metallic core, and the strong-coupling electrostatics due to multivalent ions. We specifically study the intricate roles these factors play in the electrostatic stability of the model VLPs. It is shown that while the insertion of a metallic nanoparticle by itself can produce negative, inward-directed, pressure on the outer shell, addition of only a small amount of multivalent counterions can robustly engender negative pressures, enhancing the VLP stability across a wide range of values for the system parameters.

Biomineralization improves the thermostability of foot-and-mouth disease virus-like particles and the protective immune response induced.

Virus-like particles (VLPs) are an ideal substitute for traditionally inactivated or attenuated viruses in vaccine production. However, given the properties of their native proteins, the thermal stability of VLPs is poor. In this study, calcium mineralization was used to fabricate foot-and-mouth disease virus (FMDV) VLPs as immunogenic core-shell particles with improved thermal stability. The biomineralized VLPs were stably stored at 24 °C and 37 °C for 13 and 11 days, respectively. Animal experiments showed that the biomineralized VLPs induced specific protective immunogenic effects, even after storage at 37 °C for 7 days. The biomineralized VLPs also effectively activated dendritic cells (DCs) to express high levels of surface MHC-II, costimulatory molecules, and proinflammatory cytokines. The DCs activated by the mineralized VLPs rapidly localized to the secondary lymphoid tissues and promoted the activation of the native T-cell population. These results suggest that the biomineralization of VLPs is an effective approach to vaccine production insofar as the mineralized shell provides an adjuvant effect which improves the immunogenicity of the VLPs. Biomineralization can also confer superior heat resistance on VLPs, an advantage in vaccine production. The successful development of thermally stable, biomineralized VLPs will reduce our dependence on cold storage and delivery.

The influence of ligand charge and length on the assembly of Brome mosaic virus derived virus-like particles with magnetic core

Virus-like particles (VLPs) have sparked a great interest in the field of nanobiotechnology and nanomedicine. The introduction of superparamagnetic nanoparticles (SPIONs) as a core, provides potential use of VLPs in the hyperthermia therapy, MRI contrast agents and magnetically-powered delivery agents. Magnetite NPs also provide a significant improvement in terms of VLPs stability. Moreover employing viral structural proteins as self-assembling units has opened a new paths for targeted therapy, drug delivery systems, vaccines design, and many more. In many cases, the self-assembly of a virus strongly depends on electrostatic interactions between positively charged groups of the capsid proteins and negatively charged nucleic acid. This phenomenon imposes the negative net charge as a key requirement for the core nanoparticle. In our experiments, Brome mosaic virus (BMV) capsid proteins isolated from infected plants Hordeum vulgare were used. Superparamagnetic iron oxide nanoparticles (Fe3O4) with 15 nm in diameter were synthesized by thermal decomposition and functionalized with COOH-PEG-PL polymer or dihexadecylphosphate (DHP) in order to provide water solubility and negative charge required for the assembly. Nanoparticles were characterized by Transmission Electron Microscopy (TEM), Dynamic Light Scattering (DLS), Zeta Potential, Fourier Transformed Infrared Spectroscopy (FTIR) and Superconducting Quantum Interference Device (SQUID) magnetometry. TEM and DLS study were conducted to verify VLPs creation. This study demonstrates that the increase of negative surface charge is not a sufficient factor determining successful assembly. Additional steric interactions provided by longer ligands are crucial for the assembly of BMV SPION VLPs and may enhance the colloidal stability.

Cryo-Electron Microscopy Structure of Seneca Valley Virus Procapsid.

Seneca Valley virus (SVV), like some other members of the Picornaviridae, forms naturally occurring empty capsids, known as procapsids. Procapsids have the same antigenicity as full virions, so they present an interesting possibility for the formation of stable virus-like particles. Interestingly, although SVV is a livestock pathogen, it has also been found to preferentially infect tumor cells and is being explored for use as a therapeutic agent in the treatment of small-cell lung cancers. Here we used cryo-electron microscopy to investigate the procapsid structure and describe the transition of capsid protein VP0 to the cleaved forms of VP4 and VP2. We show that the SVV receptor binds the procapsid, as evidence of its native antigenicity. In comparing the procapsid structure to that of the full virion, we also show that a cage of RNA serves to stabilize the inside surface of the virus, thereby making it more acid stable.IMPORTANCE Viruses are extensively studied to help us understand infection and disease. One of the by-products of some virus infections are the naturally occurring empty virus capsids (containing no genome), termed procapsids, whose function remains unclear. Here we investigate the structure and formation of the procapsids of Seneca Valley virus, to better understand how they form, what causes them to form, how they behave, and how we can make use of them. One potential benefit of this work is the modification of the procapsid to develop it for targeted in vivo delivery of therapeutics or to make a stable vaccine against SVV, which could be of great interest to the agricultural industry.

Stability of virus-like particles of red-spotted grouper nervous necrosis virus in the aqueous state, and the vaccine potential of lyophilized particles

Virus-like particles (VLPs) are multi protein complexes mimicking the structural properties of the native virus. The development of freeze-dried formulations of such complex protein structures remains a challenge. Red-spotted grouper nervous necrosis virus (RGNNV) causes mass mortality in fish culture, and RGNNV VLPs have been suggested to be promising vaccine candidates. In the present study, the stability of RGNNV VLPs in the liquid state was investigated over a 4-week period, along with the influence of freeze-drying on VLP stability. RGNNV VLPs were completely degraded after one week at 37 °C followed by 3 weeks at ambient temperature, and they were partially degraded after 4 weeks at 4 °C. Therefore, the inherent stability of RGNNV VLP in an aqueous milieu is insufficient for long-term storage. When RGNNV VLPs were freeze-dried in the presence or absence of sugar stabilizers, sorbitol was found to improve VLP stability whereas mannitol reduced it. VLP preparations freeze-dried with sorbitol or without stabilizer were as immunogenic as control (non-freeze dried) VLPs, whereas VLPs freeze-dried in mannitol were less immunogenic. These results indicate that freeze-dried RGNNV VLPs have potential as vaccines.