Galactogen, a virus-like particle from slugs

Virus-like particles were isolated from three species of slugs. Further investigation showed that the preparations contained very little nucleic acid and not more than 2% protein. Colorimetric and chromatographic analyses showed that they were composed almost entirely of galactogen and glycogen.     

Characterization of protection afforded by a bivalent virus-like particle vaccine against bluetongue virus serotypes 1 and 4 in sheep

Background

Bluetongue virus (BTV) is an economically important, arthropod borne, emerging pathogen in Europe, causing disease mainly in sheep and cattle. Routine vaccination for bluetongue would require the ability to distinguish between vaccinated and infected individuals (DIVA). Current vaccines are effective but are not DIVA. Virus-like particles (VLPs) are highly immunogenic structural mimics of virus particles, that only contain a subset of the proteins present in a natural infection. VLPs therefore offer the potential for the development of DIVA compatible bluetongue vaccines.

Methodology/Principal Findings

Merino sheep were vaccinated with either monovalent BTV-1 VLPs or a bivalent mixture of BTV-1 VLPs and BTV-4 VLPs, and challenged with virulent BTV-1 or BTV-4. Animals were monitored for clinical signs, antibody responses, and viral RNA. 19/20 animals vaccinated with BTV-1 VLPs either alone or in combination with BTV-4 VLPs developed neutralizing antibodies to BTV-1, and group specific antibodies to BTV VP7. The one animal that showed no detectable neutralizing antibodies, or group specific antibodies, had detectable viral RNA following challenge but did not display any clinical signs on challenge with virulent BTV-1. In contrast, all control animals'' demonstrated classical clinical signs for bluetongue on challenge with the same virus. Six animals were vaccinated with bivalent vaccine and challenged with virulent BTV-4, two of these animals had detectable viral levels of viral RNA, and one of these showed clinical signs consistent with BTV infection and died.

Conclusions

There is good evidence that BTV-1 VLPs delivered as monovalent or bivalent immunogen protect from bluetongue disease on challenge with virulent BTV-1. However, it is possible that there is some interference in protective response for BTV-4 in the bivalent BTV-1 and BTV-4 VLP vaccine. This raises the question of whether all combinations of bivalent BTV vaccines are possible, or if immunodominance of particular serotypes could interfere with vaccine efficacy.     

Hepatitis C virus-like particle morphogenesis

Although much is known about the hepatitis C virus (HCV) genome, first cloned in 1989, little is known about HCV structure and assembly due to the lack of an efficient in vitro culture system for HCV. Using a recombinant Semliki forest virus replicon expressing genes encoding HCV structural proteins, we observed for the first time the assembly of these proteins into HCV-like particles in mammalian cells. This system opens up new possibilities for the investigation of viral morphogenesis and virus-host cell interactions.     

Engineered mutations change the structure and stability of a virus-like particle

The single-coat protein (CP) of bacteriophage Qβ self-assembles into T = 3 icosahedral virus-like particles (VLPs), of interest for a wide range of applications. These VLPs are very stable, but identification of the specific molecular determinants of this stability is lacking. To investigate these determinants along with manipulations that confer more capabilities to our VLP material, we manipulated the CP primary structure to test the importance of various putative stabilizing interactions. Optimization of a procedure to incorporate fused CP subunits allowed for good control over the average number of covalent dimers in each VLP. We confirmed that the disulfide linkages are the most important stabilizing elements for the capsid and that acidic conditions significantly enhance the resistance of VLPs to thermal degradation. Interdimer interactions were found to be less important for VLP assembly than intradimer interactions. Finally, a single point mutation in the CP resulted in a population of smaller VLPs in three distinct structural forms.     

Structure and assembly of a T=1 virus-like particle in BK polyomavirus

In polyomaviruses the pentameric capsomers are interlinked by the long C-terminal arm of the structural protein VP1. The T=7 icosahedral structure of these viruses is possible due to an intriguing adaptability of this linker arm to the different local environments in the capsid. To explore the assembly process, we have compared the structure of two virus-like particles (VLPs) formed, as we found, in a calcium-dependent manner by the VP1 protein of human polyomavirus BK. The structures were determined using electron cryomicroscopy (cryo-EM), and the three-dimensional reconstructions were interpreted by atomic modeling. In the small VP1 particle, 26.4 nm in diameter, the pentameric capsomers form an icosahedral T=1 surface lattice with meeting densities at the threefold axes that interlinked three capsomers. In the larger particle, 50.6 nm in diameter, the capsomers form a T=7 icosahedral shell with three unique contacts. A folding model of the BKV VP1 protein was obtained by alignment with the VP1 protein of simian virus 40 (SV40). The model fitted well into the cryo-EM density of the T=7 particle. However, residues 297 to 362 of the C-terminal arm had to be remodeled to accommodate the higher curvature of the T=1 particle. The loops, before and after the C-terminal short helix, were shown to provide the hinges that allowed curvature variation in the particle shell. The meeting densities seen at the threefold axes in the T=1 particle were consistent with the triple-helix interlinking contact at the local threefold axes in the T=7 structure.     

Characterization of porcine circovirus type 2 (PCV2) capsid particle assembly and its application to virus-like particle vaccine development

Porcine circovirus type 2 (PCV2) is the primary causative agent of porcine circovirus-associated diseases in pigs. The sole structural capsid protein of PCV2, Cap, consists of major antigenic domains, but little is known about the assembly of capsid particles. The purpose of this study is to produce a large amount of Cap protein using Escherichia coli expression system for further studying the essential sequences contributing to formation of particles. By using codon optimization of rare arginine codons near the 5'-end of the cap gene for E. coli, a full-length Cap without any fusion tag recombinant protein (Cap1-233) was expressed and proceeded to form virus-like particles (VLPs) in normal Cap appearance that resembled the authentic PCV2 capsid. The N-terminal deletion mutant (Cap51-233) deleted the nuclear localization signal (NLS) domain, while the internal deletion mutant (CapΔ51-103) deleted a likely dimerization domain that failed to form VLPs. The unique Cys108 substitution mutant (CapC/S) exhibited most irregular aggregates, and only few VLPs were formed. These results suggest that the N-terminal region within the residues 1 to 103 possessing the NLS and dimerization domains are essential for self-assembly of stable Cap VLPs, and the unique Cys108 plays an important role in the integrity of VLPs. The immunogenicity of PCV2 VLPs was further evaluated by immunization of pigs followed by challenge infection. The Cap1-233-immunized pigs demonstrated specific antibody immune responses and are prevented from PCV2 challenge, thus implying its potential use for a VLP-based PCV2 vaccine.     

流感病毒样颗粒疫苗研究进展

流行性感冒(流感)是由正粘病毒科的负链RNA流感病毒引起的一种上呼吸道急性传染病,它传染性强、传播快、潜伏期短、发病率高,严重影响着人类的健康和全球经济的发展,对老年人和婴幼儿危害尤其大。在人类历史上曾经历过多次流感大流     

Characterization of a latent virus-like infection of symbiotic zooxanthellae

A latent virus-like agent, which we designated zooxanthella filamentous virus 1 (ZFV1), was isolated from Symbiodinium sp. strain CCMP 2465 and characterized. Transmission electron microscopy and analytical flow cytometry revealed the presence of a new group of distinctive filamentous virus-like particles after exposure of the zooxanthellae to UV light. Examination of thin sections of the zooxanthellae revealed the formation and proliferation of filamentous virus-like particles in the UV-induced cells. Assessment of Symbiodinium sp. cultures was used here as a model to show the effects of UV irradiance and induction of potential latent viruses. The unique host-virus system described here provides insight into the role of latent infections in zooxanthellae through environmentally regulated viral induction mechanisms.     

Production of CCHF virus-like particle by a baculovirus-insect cell expression system

Crimean-Congo Haemorrhagic Fever Virus (CCHFV) is a tick-born virus of the Nairovirus genus within the Bunyaviridae family, which is widespread and causes high fatality. The nucleocapsid of CCHFV is comprised of N proteins that are encoded by the S segment. In this research, the N protein of CCHFV was expressed in insect cells using a recombinant baculovirus. Under an electron microscope, Virus-Like Particles (VLPs) with various size and morphology were observed in cytoplasmic vesicles in the infected cells. Sucrose-gradient purification of the cell lysate indicated that the VLPs were mainly located in the upper fraction after ultracentrifugation, which was confirmed by Western blot analysis and immuno-electron microscopy (IEM).     

Synthetic biology for bioengineering virus-like particle vaccines

Vaccination is the most effective method of disease prevention and control. Many viruses and bacteria that once caused catastrophic pandemics (e.g., smallpox, poliomyelitis, measles, and diphtheria) are either eradicated or effectively controlled through routine vaccination programs. Nonetheless, vaccine manufacturing remains incredibly challenging. Viruses exhibiting high antigenic diversity and high mutation rates cannot be fairly contested using traditional vaccine production methods and complexities surrounding the manufacturing processes, which impose significant limitations. Virus-like particles (VLPs) are recombinantly produced viral structures that exhibit immunoprotective traits of native viruses but are noninfectious. Several VLPs that compositionally match a given natural virus have been developed and licensed as vaccines. Expansively, a plethora of studies now confirms that VLPs can be designed to safely present heterologous antigens from a variety of pathogens unrelated to the chosen carrier VLPs. Owing to this design versatility, VLPs offer technological opportunities to modernize vaccine supply and disease response through rational bioengineering. These opportunities are greatly enhanced with the application of synthetic biology, the redesign and construction of novel biological entities. This review outlines how synthetic biology is currently applied to engineer VLP functions and manufacturing process. Current and developing technologies for the identification of novel target-specific antigens and their usefulness for rational engineering of VLP functions (e.g., presentation of structurally diverse antigens, enhanced antigen immunogenicity, and improved vaccine stability) are described. When applied to manufacturing processes, synthetic biology approaches can also overcome specific challenges in VLP vaccine production. Finally, we address several challenges and benefits associated with the translation of VLP vaccine development into the industry.     

The coming of age of virus-like particle vaccines

Virus-like particles are supra-molecular assemblages, usually icosahedral or rod-like structures. They incorporate key immunologic features of viruses which include repetitive surfaces, particulate structures and induction of innate immunity through activation of pathogen-associated molecular-pattern recognition receptors. They carry no replicative genetic information and can be produced recombinantly in large scale. Virus-like particles thus represent a safe and effective vaccine platform for inducing potent B- and T-cell responses. In addition to being effective vaccines against the corresponding virus from which they are derived, virus-like particles can also be used to present foreign epitopes to the immune system. This can be achieved by genetic fusion or chemical conjugation. This technological innovation has greatly broadened the scope of their use, from immunizing against microbial pathogens to immunotherapy for chronic diseases. Towards this end, virus-like particles have been used to induce autoantibodies to disease-associated self-molecules involved in chronic diseases, such as hypertension and Alzheimer's disease. The recognition of the potent immunogenicity and commercial potential for virus-like particles has greatly accelerated research and development activities. During the last decade, two prophylactic virus-like particle vaccines have been registered for human use, while another 12 vaccines entered clinical development.     

Biochemical and physiological studies of the yeast virus-like particle.

A study was made of the virus-like particle (VLP) of Saccharomyces cerevisiae S7. This strain contains elevated amounts of P1 double-stranded ribonucleic acid (dsRNA) but no P2 dsRNA. The amount of dsRNA contained in cells grown on a fermentable carbon source (glucose) was compared with that in cells grown on a nonfermentable carbon source (ethanol). It was found that ethanol-grown cells contain higher levels of dsRNA than glucose-grown cells. In the former, the amount of dsRNA increased during the logarithmic phase of growth, whereas in the latter it increased during the transition from the logarithmic to the stationary phase. A method was devised to isolate VLPs from these cells by using CsCl gradients, and the yield was assessed by monitoring the recovery of dsRNA. Three proteins were found to be tightly associated with these particles. They have molecular weights of 75,000, 53,000, and 37,000. Together they account for almost all of the coding capacity of the P1 dsRNA that the VLP contains.     

Quantitative analysis of virus-like particle size and distribution by field-flow fractionation

Asymmetric flow field-flow fractionation (AFFFF) coupled with multiple-angle light scattering (MALS) is a powerful technique showing potential for the analysis of pharmaceutically-relevant virus-like particles (VLPs). A lack of published methods, and concerns that membrane adsorption during sample fractionation may cause sample aggregation, have limited widespread acceptance. Here we report a reliable optimized method for VLP analysis using AFFFF-MALS, and benchmark it against dynamic light scattering (DLS) and transmission electron microscopy (TEM). By comparing chemically identical VLPs having very different quaternary structure, sourced from both bacteria and insect cells, we show that optimized AFFFF analysis does not cause significant aggregation, and that accurate size and distribution information can be obtained for heterogeneous samples in a way not possible with TEM and DLS. Optimized AFFFF thus provides a quantitative way to monitor batch consistency for new vaccine products, and rapidly provides unique information on the whole population of particles within a sample.     

Self-assembly of virus-like particles of canine parvovirus capsid protein expressed from Escherichia coli and application as virus-like particle vaccine

Canine parvovirus disease is an acute infectious disease caused by canine parvovirus (CPV). Current commercial vaccines are mainly attenuated and inactivated; as such, problems concerning safety may occur. To resolve this problem, researchers developed virus-like particles (VLPs) as biological nanoparticles resembling natural virions and showing high bio-safety. This property allows the use of VLPs for vaccine development and mechanism studies of viral infections. Tissue-specific drug delivery also employs VLPs as biological nanomaterials. Therefore, VLPs derived from CPV have a great potential in medicine and diagnostics. In this study, small ubiquitin-like modifier (SUMO) fusion motif was utilized to express a whole, naturalVP2 protein of CPV in Escherichia coli. After the cleavage of the fusion motif, the CPV VP2 protein has self-assembled into VLPs. The VLPs had a size and shape that resembled the authentic virus capsid. However, the self-assembly efficiency of VLPs can be affected by different pH levels and ionic strengths. The mice vaccinated subcutaneously with CPV VLPs and CPV-specific immune responses were compared with those immunized with the natural virus. This result showed that VLPs can effectively induce anti-CPV specific antibody and lymphocyte proliferation as a whole virus. This result further suggested that the antigen epitope of CPV was correctly present on VLPs, thereby showing the potential application of a VLP-based CPV vaccine.     

Immunogenicity and virus-like particle formation of rotavirus capsid proteins produced in transgenic plants

The human pathogen, group A rotavirus, is the most prevalent cause of acute infantile and pediatric gastroenteritis worldwide, especially in developing countries. There is an urgent demand for safer, more effective and cheaper vaccines against rotavirus infection. Plant-derived antigens may provide an exclusive way to produce economical subunit vaccines. Virus-like particles, constituting viral capsid proteins without viral nucleic acids, are considered a far safer candidate compared with live attenuated viral vaccines. In this study, the rotavirus capsid proteins VP2, VP6 and VP7 were co-expressed in transgenic tobacco plants, and their expression levels, formation of rotavirus-like particles (RV VLPs) and immunogenicity were extensively studied. Quantitative real-time RT-PCR and Western blot analysis revealed that the expression level of vp6 was the highest while vp7 was expressed at the lowest levels. The RV VLPs were purified from transgenic tobacco plants and analyzed by electron microscopy and Western blot. Results indicated that the plant-derived VP2, VP6 and VP7 proteins self-assembled into 2/6 or 2/6/7 RV VLPs with a diameter of 60–80 nm. When orally delivered into mice with cholera toxin as an adjuvant, the total soluble protein extracted from transgenic tobacco plants induced rotavirus-specific antibodies comparable with those of attenuated rotavirus vaccines, while VP 2/6/7 induced higher serum IgG and fecal IgA titers compared with VP 2/6.     

Integrated process optimization: lessons from retrovirus and virus-like particle production

The optimization of production and purification processes is usually approached by engineers from a strictly biotechnological point of view. The present paper envisages the definition and application of an optimization model that takes into account the impact of both biological and technological issues upon the optimization protocols and strategies. For this purpose, the optimization of three analogous but different systems comprising animal cell growth and bioparticle production is presented. These systems were: human immunodeficiency 1 (HIV-1) and porcine parvovirus (PPV) virus-like particles (VLPs) produced in insect cells and retrovirus produced in mammalian cells. For the systematization of the optimization process four levels of optimization were defined-product, technology, design and integration. In this paper, the limits of each of the optimization levels defined are discussed by applying the concept to the systems described. This analysis leads to decisions regarding the production of VLPs and retrovirus as well as on the points relevant for further process development. Finally, the definition of the objective function or performance index, the possible strategies and tools for bioprocess optimization are described. Although developed from the three described processes, this approach can, based on the recent literature evidence reviewed here, be applied more universally for the process development of complex biopharmaceuticals.     

Low temperature-dependent salmonid alphavirus glycoprotein processing and recombinant virus-like particle formation

Pancreas disease (PD) and sleeping disease (SD) are important viral scourges in aquaculture of Atlantic salmon and rainbow trout. The etiological agent of PD and SD is salmonid alphavirus (SAV), an unusual member of the Togaviridae (genus Alphavirus). SAV replicates at lower temperatures in fish. Outbreaks of SAV are associated with large economic losses of ~17 to 50 million $/year. Current control strategies rely on vaccination with inactivated virus formulations that are cumbersome to obtain and have intrinsic safety risks. In this research we were able to obtain non-infectious virus-like particles (VLPs) of SAV via expression of recombinant baculoviruses encoding SAV capsid protein and two major immunodominant viral glycoproteins, E1 and E2 in Spodoptera frugiperda Sf9 insect cells. However, this was only achieved when a temperature shift from 27°C to lower temperatures was applied. At 27°C, precursor E2 (PE2) was misfolded and not processed by host furin into mature E2. Hence, E2 was detected neither on the surface of infected cells nor as VLPs in the culture fluid. However, when temperatures during protein expression were lowered, PE2 was processed into mature E2 in a temperature-dependent manner and VLPs were abundantly produced. So, temperature shift-down during synthesis is a prerequisite for correct SAV glycoprotein processing and recombinant VLP production.     

Intranasal vaccination with murabutide enhances humoral and mucosal immune responses to a virus-like particle vaccine

Murabutide (MB) is a synthetic immunomodulator recognized by the nucleotide-binding oligomerization domain-containing protein 2 (NOD2) receptor on mammalian cells. MB has previously been approved for testing in multiple human clinical trials to determine its value as an antiviral therapeutic, and as an adjuvant for injected vaccines. We have found a new use for this immunomodulator; it functions as a mucosal adjuvant that enhances immunogenicity of virus-like particles (VLP) administered intranasally. MB enhanced Norwalk virus (NV) VLP-specific IgG systemically and IgA production at distal mucosal sites following intranasal (IN) vaccination. A dose escalation study identified 100 μg as the optimal MB dosage in mice, based on the magnitude of VLP-specific IgG, IgG1, IgG2a and IgA production in serum and VLP-specific IgA production at distal mucosal sites. IN vaccination using VLP with MB was compared to IN delivery VLP with cholera toxin (CT) or gardiquimod (GARD) and to parenteral VLP delivery with alum; the MB groups were equivalent to CT and GARD and superior to alum in inducing mucosal immune responses and stimulated equivalent systemic VLP-specific antibodies. These data support the further testing of MB as a potent mucosal adjuvant for inducing robust and durable antibody responses to non-replicating subunit vaccines.     

Intrinsic fluorescence as an analytical probe of virus-like particle assembly and maturation

The assembly and maturation of the human papillomavirus (HPV) virus-like particle (VLP) has been monitored by measuring the intrinsic fluorescence intensity using excitation at 290 nm and emission at 350 nm. The assay was validated to eliminate error due to photo-bleaching, adsorption, and precipitation. Intrinsic fluorescence intensity dropped during both assembly and maturation phases. The decrease during assembly had a second-order dependence on capsomere concentration, as previously observed using light scattering. During post-assembly structural modification the decrease had a first-order dependence on capsomere concentration. Intrinsic fluorescence spectroscopy complements light scattering methodologies for monitoring assembly and enables kinetics of maturation to be observed. The role of environmental factors such as the presence of oxidized glutathione in facilitation of faster and more complete maturation was monitored in real time. Intrinsic fluorescence is a rugged methodology that could be applied to monitoring VLP assembly and maturation unit operations during HPV vaccine manufacturing.