Evaluation of electrospray differential mobility analysis for virus particle analysis: Potential applications for biomanufacturing.
ABSTRACT: The technique of electrospray differential mobility analysis (ES-DMA) was examined as a potential potency assay for routine virus particle analysis in biomanufacturing environments (e.g., evaluation of vaccines and gene delivery products for lot release) in the context of the International Committee of Harmonisation (ICH) Q2 guidelines. ES-DMA is a rapid particle sizing method capable of characterizing certain aspects of the structure (such as capsid proteins) and obtaining complete size distributions of viruses and virus-like particles. It was shown that ES-DMA can distinguish intact virus particles from degraded particles and measure the concentration of virus particles when calibrated with nanoparticles of known concentration. The technique has a measurement uncertainty of ?20%, is linear over nearly 3 orders of magnitude, and has a lower limit of detection of ?10(9)particles/mL. This quantitative assay was demonstrated for non-enveloped viruses. It is expected that ES-DMA will be a useful method for applications involving production and quality control of vaccines and gene therapy vectors for human use.
Project description:Collagen is a natural polymer found abundantly in the extracellular matrix (ECM). It is easily extracted from a variety of sources and exhibits excellent biological properties such as biocompatibility and weak antigenicity. Additionally, different processes allow control of physical and chemical properties such as mechanical stiffness, viscosity and biodegradability. Moreover, various additive biomanufacturing technology has enabled layer-by-layer construction of complex structures to support biological function. Additive biomanufacturing has expanded the use of collagen biomaterial in various regenerative medicine and disease modelling application (e.g., skin, bone and cornea). Currently, regulatory hurdles in translating collagen biomaterials still remain. Additive biomanufacturing may help to overcome such hurdles commercializing collagen biomaterials and fulfill its potential for biomedicine.
Project description:Recombinant virus-like particle-based vaccines are composed of viral structural proteins and mimic authentic native viruses but are devoid of viral genetic materials. They are the active components in highly safe and effective vaccines for the prevention of infectious diseases. Several expression systems have been used for virus-like particle production, ranging from Escherichia coli to mammalian cell lines. The prokaryotic expression system, especially Escherichia coli, is the preferred expression host for producing vaccines for global use. Hecolin, the first licensed virus-like particle vaccine derived from Escherichia coli, has been demonstrated to possess good safety and high efficacy. In this review, we focus on Escherichia coli-derived virus-like particle based vaccines and vaccine candidates that are used for prevention (immunization against microbial pathogens) or disease treatment (directed against cancer or non-infectious diseases). The native-like spatial or higher-order structure is essential for the function of virus-like particles. Thus, the tool box for analyzing the key physicochemical, biochemical and functional attributes of purified virus-like particles will also be discussed. In summary, the Escherichia coli expression system has great potentials for producing a range of proteins with self-assembling properties to be used as vaccine antigens given the proper epitopes were preserved when compared to those in the native pathogens or disease-related target molecules.
Project description:The achievements of cell-based therapeutics have galvanized efforts to bring cell therapies to the market. To address the demands of the clinical and eventual commercial-scale production of cells, and with the increasing generation of large clinical datasets from chimeric antigen receptor T-cell immunotherapy, from transplants of engineered haematopoietic stem cells and from other promising cell therapies, an emphasis on biomanufacturing requirements becomes necessary. Robust infrastructure should address current limitations in cell harvesting, expansion, manipulation, purification, preservation and formulation, ultimately leading to successful therapy administration to patients at an acceptable cost. In this Review, we highlight case examples of cutting-edge bioprocessing technologies that improve biomanufacturing efficiency for cell therapies approaching clinical use.
Project description:Bluetongue virus (BTV) causes severe disease in domestic and wild ruminants, and has recently caused several outbreaks in Europe. Current vaccines include live-attenuated and inactivated viruses; while these are effective, there is risk of reversion to virulence by mutation or reassortment with wild type viruses. Subunit or virus-like particle (VLP) vaccines are safer options: VLP vaccines produced in insect cells by expression of the four BTV capsid proteins are protective against challenge; however, this is a costly production method. We investigated production of BTV VLPs in plants via Agrobacterium-mediated transient expression, an inexpensive production system very well suited to developing country use. Leaves infiltrated with recombinant pEAQ-HT vectors separately encoding the four BTV-8 capsid proteins produced more proteins than recombinant pTRA vectors. Plant expression using the pEAQ-HT vector resulted in both BTV-8 core-like particles (CLPs) and VLPs; differentially controlling the concentration of infiltrated bacteria significantly influenced yield of the VLPs. In situ localisation of assembled particles was investigated by using transmission electron microscopy (TEM) and it was shown that a mixed population of core-like particles (CLPs, consisting of VP3 and VP7) and VLPs were present as paracrystalline arrays in the cytoplasm of plant cells co-expressing all four capsid proteins.
Project description:A cavity ring-down spectrometer and condensation particle counter were used to investigate the limitations in the separation of singly and multiply charged aerosol particles by a tandem differential mobility analyzer (DMA) and aerosol particle mass analyzer (APM). The impact of particle polydispersity and morphology was investigated using three materials: nearly-monodisperse polystyrene latex nanospheres (PSL); polydisperse, nearly-spherical ammonium sulfate (AS) and polydisperse lacey fractal soot agglomerates. PSL and AS particles were easily resolved as a function of charge. For fresh soot, the presence of multiply charged particles severely affects the isolation of the singly charged particles. In cases where the DMA-APM was unable to fully resolve the singly charged particles of interest, the peak mass deviated by up to 13 % leading to errors in the mass specific extinction cross section of over 100 %. For measurements of non-spherical particles, non-symmetrical distributions of concentration as a function of mass were a sign of the presence of multiply charged particles. Under these conditions, the effects of multiply charged particles can be reduced by using a second charge neutralizer after the DMA and prior to the APM. Dilution of the aerosol stream serves to decrease the total number concentration of particles and does not remove the contributions of multiply charged particles.
Project description:Here we generate silk-elastin-like protein (SELP) polymeric nanoparticles and demonstrate precise control over their dimensions using an electrospray differential mobility analyzer (ES-DMA). Electrospray produces droplets encompassing several polymer strands. Evaporation ensues, leading polymer strands to accumulate at the droplet interface, forming a hollow nanoparticle. The resulting nanoparticle size distributions, which govern particle yield, depend on buffer concentration to the -1/3 power, polymer concentration to the 1/3 power, and ratio of silk-to-elastin blocks. Three recombinantly tuned ratios of 8:16, 4:8, and 4:16, respectively named SELP-815K, SELP-47K, and SELP-415K, are employed, with the latter ratio resulting in a thinner shell and larger diameter for the nanoparticles than the former. The DMA narrows the size distribution by electrostatically classifying the aerosolized nanoparticles. These highly uniform nanoparticles have variations of 1.2 and 1.4 nm for 24.0 and 36.0 nm particles, respectively. Transmission electron microscopy reveals the nanoparticles to be faceted, as a buckling instability releases compression energy arising from evaporation after the shell has formed by bending it. A thermodynamic equilibrium exists between compression and bending energies, where the facet length is half the particle diameter, in agreement with experiments. Rod-like particles also formed from polymer-stabilized filaments when the viscous length exceeds the jet radius at higher solution viscosities. The unusual uniformity in composition and dimension indicates the potential of these nanoparticles to deliver bioactive and imaging agents.
Project description:Viruses in the Flaviviridae family are important human and animal pathogens that impose serious threats to global public health. This family of viruses includes emerging and re-emerging viruses, most of which are transmitted by infected mosquito or tick bites. Currently, there is no protective vaccine or effective antiviral treatment against the majority of these viruses, and due to their growing spread, several strategies have been employed to manufacture prophylactic vaccines against these infectious agents including virus-like particle (VLP) subunit vaccines. VLPs are genomeless viral particles that resemble authentic viruses and contain critical repetitive conformational structures on their surface that can trigger the induction of both humoral and cellular responses, making them safe and ideal vaccine candidates against these viruses. In this review, we focus on the potential of the VLP platform in the current vaccine development against the medically important viruses in the Flaviviridae family.
Project description:Colchicine is one of the oldest plant-based medicines used to treat gout and one of the most important alkaloid-based antimitotic drugs with anticancer potential, which is commercially extracted from Gloriosa superba. Clinical trials suggest that colchicine medication could prevent atrial fibrillation recurrence after cardiac surgery. In addition, therapeutic colchicine is undergoing clinical trials to treat non-diabetic metabolic syndrome and diabetic nephropathy. However, the industrial-scale biomanufacturing of colchicine have not yet been established. Clearly, further studies on detailed biorhizome-specific transcriptome analysis, gene expression, and candidate gene validation are required before uncover the mechanism of colchicine biosynthesis and biorhizome-based colchicine biomanufacturing. Annotation of 32312 assembled multiple-tissues transcripts of G. superba represented 15088 unigenes in known plant specific gene ontology. This could help understanding colchicine biosynthesis in G. superba. This review highlights the biorhizomes, rhizome specific genes or gene what expressed with high level in rhizomes, and deep fluid dynamics in a bioreactor specifically for the biomanufacture of colchicine.
Project description:This study addresses the role of Ebola virus (EBOV) specific infectivity in virulence. Filoviruses are highly lethal, enveloped, single-stranded negative-sense RNA viruses that can cause hemorrhagic fever. No approved vaccines or therapies exist for filovirus infections, and infectious virus must be handled in maximum containment. Efficacy testing of countermeasures, in addition to investigations of pathogenicity and immune response, often requires a well-characterized animal model. For EBOV, an obstacle in performing accurate disease modeling is a poor understanding of what constitutes an infectious dose in animal models. One well-recognized consequence of viral passage in cell culture is a change in specific infectivity, often measured as a particle-to-PFU ratio. Here, we report that serial passages of EBOV in cell culture resulted in a decrease in particle-to-PFU ratio. Notably, this correlated with decreased potency in a lethal cynomolgus macaque (Macaca fascicularis) model of infection; animals were infected with the same viral dose as determined by plaque assay, but animals that received more virus particles exhibited increased disease. This suggests that some particles are unable to form a plaque in a cell culture assay but are able to result in lethal disease in vivo. These results have a significant impact on how future studies are designed to model EBOV disease and test countermeasures.Ebola virus (EBOV) can cause severe hemorrhagic disease with a high case-fatality rate, and there are no approved vaccines or therapies. Specific infectivity can be considered the total number of viral particles per PFU, and its impact on disease is poorly understood. In stocks of most mammalian viruses, there are particles that are unable to complete an infectious cycle or unable to cause cell pathology in cultured cells. We asked if these particles cause disease in nonhuman primates by infecting monkeys with equal infectious doses of genetically identical stocks possessing either high or low specific infectivities. Interestingly, some particles that did not yield plaques in cell culture assays were able to result in lethal disease in vivo. Furthermore, the number of PFU needed to induce lethal disease in animals was very low. Our results have a significant impact on how future studies are designed to model EBOV disease and test countermeasures.
Project description:Successful live attenuated vaccines mimic natural exposure to pathogens without causing disease and have been successful against several viruses. However, safety concerns prevent the development of attenuated human immunodeficiency virus (HIV) as a vaccine candidate. If a safe, replicating virus vaccine could be developed, it might have the potential to offer significant protection against HIV infection and disease. Described here is the development of a novel self-replicating chimeric virus vaccine candidate that is designed to provide natural exposure to a lentivirus-like particle and to incorporate the properties of a live attenuated virus vaccine without the inherent safety issues associated with attenuated lentiviruses. The genome from the alphavirus Venezuelan equine encephalitis virus (VEE) was modified to express SHIV89.6P genes encoding the structural proteins Gag and Env. Expression of Gag and Env from VEE RNA in primate cells led to the assembly of particles that morphologically and functionally resembled lentivirus virions and that incorporated alphavirus RNA. Infection of CD4? cells with chimeric lentivirus-like particles was specific and productive, resulting in RNA replication, expression of Gag and Env, and generation of progeny chimeric particles. Further genome modifications designed to enhance encapsidation of the chimeric virus genome and to express an attenuated simian immunodeficiency virus (SIV) protease for particle maturation improved the ability of chimeric lentivirus-like particles to propagate in cell culture. This study provides proof of concept for the feasibility of creating chimeric virus genomes that express lentivirus structural proteins and assemble into infectious particles for presentation of lentivirus immunogens in their native and functional conformation.