RESUMO
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.
Assuntos
Bioengenharia/métodos , Vacinas de Partículas Semelhantes a Vírus , Animais , Biologia Computacional/métodos , Humanos , Modelos Moleculares , Biologia Sintética/métodos , Vacinas de Partículas Semelhantes a Vírus/química , Vacinas de Partículas Semelhantes a Vírus/genética , Vacinas de Partículas Semelhantes a Vírus/imunologiaRESUMO
Development of antifouling films which selectively capture or target proteins of interest is essential for controlling interactions at the "bio/nano" interface. However, in order to synthesize biofunctional films from synthetic polymers that incorporate chemical "motifs" for surface immobilization, antifouling, and oriented biomolecule attachment, multiple reaction steps need to be carried out at the solid/liquid interface. EKx is a zwitterionic peptide that has previously been shown to have excellent antifouling properties. In this study, we recombinantly expressed EKx peptides and genetically encoded both surface attachment and antibody-binding motifs, before characterizing the resultant biopolymers by traditional methods. These peptides were then immobilized to organosilica nanoparticles for binding IgG, and subsequently capturing dengue NS1 as a model antigen from serum-containing solution. We found that a mixed layer of a short peptide (4.9 kDa) "backfilled" with a longer peptide terminated with an IgG-binding Z-domain (18 kDa) demonstrated selective capture of dengue NS1 protein down to â¼10 ng mL-1 in either PBS or 20% serum.
Assuntos
Incrustação Biológica/prevenção & controle , Imunoglobulina G/metabolismo , Peptídeos/metabolismo , Proteínas Recombinantes/metabolismo , Vírus da Dengue/química , Escherichia coli/genética , Proteínas Imobilizadas/genética , Proteínas Imobilizadas/metabolismo , Imunoglobulina G/química , Nanopartículas/química , Peptídeos/genética , Ligação Proteica , Domínios Proteicos , Engenharia de Proteínas/métodos , Proteínas Recombinantes/genética , Dióxido de Silício/química , Proteínas não Estruturais Virais/metabolismoRESUMO
Baculovirus-insect cell expression system has become one of the most widely used eukaryotic expression systems for heterologous protein production in many laboratories. The availability of robust insect cell lines, serum-free media, a range of vectors and commercially-packaged kits have supported the demand for maximizing the exploitation of the baculovirus-insect cell expression system. Naturally, this resulted in varied strategies adopted by different laboratories to optimize protein production. Most laboratories have preference in using either the E. coli transposition-based recombination bacmid technology (e.g. Bac-to-Bac®) or homologous recombination transfection within insect cells (e.g. flashBAC™). Limited data is presented in the literature to benchmark the protocols used for these baculovirus vectors to facilitate the selection of a system for optimal production of target proteins. Taking advantage of the Protein Production and Purification Partnership in Europe (P4EU) scientific network, a benchmarking initiative was designed to compare the diverse protocols established in thirteen individual laboratories. This benchmarking initiative compared the expression of four selected intracellular proteins (mouse Dicer-2, 204â¯kDa; human ABL1 wildtype, 126â¯kDa; human FMRP, 68â¯kDa; viral vNS1-H1, 76â¯kDa). Here, we present the expression and purification results on these proteins and highlight the significant differences in expression yields obtained using different commercially-packaged baculovirus vectors. The highest expression level for difficult-to-express intracellular protein candidates were observed with the EmBacY baculovirus vector system.
Assuntos
Baculoviridae/genética , Vetores Genéticos/genética , Proteínas Recombinantes/metabolismo , Animais , Linhagem Celular , Escherichia coli/genética , Escherichia coli/metabolismo , Proteína do X Frágil da Deficiência Intelectual/genética , Proteína do X Frágil da Deficiência Intelectual/metabolismo , Humanos , Camundongos , Proteínas Proto-Oncogênicas c-abl/genética , Proteínas Proto-Oncogênicas c-abl/metabolismo , Proteínas Recombinantes/genética , Ribonuclease III/genética , Ribonuclease III/metabolismo , Células Sf9RESUMO
Rapid advances in intensifying upstream processes for biologics production have left downstream processing as a bottleneck in the manufacturing scheme. Biomanufacturers are pursuing continuous downstream process development to increase efficiency and flexibility, reduce footprint and cost of goods, and improve product consistency and quality. Even after successful laboratory trials, the implementation of a continuous process at manufacturing scale is not easy to achieve. This paper reviews specific challenges in converting each downstream unit operation to a continuous mode. Key elements of developing practical strategies for overcoming these challenges are detailed. These include equipment valve complexity, favorable column aspect ratio, protein-A resin selection, quantitative assessment of chromatogram peak size and shape, holistic process characterization approach, and a customized process economic evaluation. Overall, this study provides a comprehensive review of current trends and the path forward for implementing continuous downstream processing at the manufacturing scale.
Assuntos
Anticorpos Monoclonais , Reatores Biológicos , Biotecnologia , Animais , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Monoclonais/metabolismo , Biotecnologia/métodos , Biotecnologia/normas , Biotecnologia/tendências , Cromatografia , Humanos , Membranas ArtificiaisRESUMO
A high global burden of rotavirus disease and the unresolved challenges with the marketed rotavirus vaccines, particularly in the developing world, have ignited efforts to develop virus-like particle (VLP) vaccines for rotavirus. While rotavirus-like particles comprising multiple viral proteins can be difficult to process, modular VLPs presenting rotavirus antigenic modules are promising alternatives in reducing process complexity and cost. In this study, integrated molecular and bioprocess engineering approaches were used to simplify the production of modular murine polyomavirus capsomeres and VLPs presenting a rotavirus 18 kDa VP8* antigen. A single construct was generated for dual expression of non-tagged murine polyomavirus capsid protein VP1 and modular VP1 inserted with VP8*, for co-expression in Escherichia coli. Co-expressed proteins assembled into pentameric capsomeres in E. coli. A selective salting-out precipitation and a polishing size exclusion chromatography step allowed the recovery of stable modular capsomeres from cell lysates at high purity, and modular capsomeres were successfully translated into modular VLPs when assembled in vitro. Immunogenicity study in mice showed that modular capsomeres and VLPs induced high levels of VP8*-specific antibodies. Our results demonstrate that a multipronged synthetic biology approach combining molecular and bioprocess engineering enabled simple and low-cost production of highly immunogenic modular capsomeres and VLPs presenting conformational VP8* antigenic modules. This strategy potentially provides a cost-effective production route for modular capsomere and VLP vaccines against rotavirus, highly suitable to manufacturing economics for the developing world. Biotechnol. Bioeng. 2017;114: 397-406. © 2016 Wiley Periodicals, Inc.
Assuntos
Antígenos Virais/genética , Bioengenharia/métodos , Proteínas Recombinantes de Fusão/genética , Rotavirus/imunologia , Vacinas de Partículas Semelhantes a Vírus/genética , Animais , Anticorpos Antivirais/sangue , Antígenos Virais/química , Antígenos Virais/imunologia , Antígenos Virais/metabolismo , Proteínas do Capsídeo/genética , Escherichia coli/genética , Camundongos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Rotavirus/genética , Vacinas de Partículas Semelhantes a Vírus/química , Vacinas de Partículas Semelhantes a Vírus/imunologia , Vacinas de Partículas Semelhantes a Vírus/metabolismoRESUMO
Enterovirus 71 (EV71) and Coxsackievirus A16 (CVA16) are two viruses commonly responsible for hand, foot and mouth disease (HFMD) in children. The lack of prophylactic or therapeutic measures against HFMD is a major public health concern. Insect cell-based EV71 and CVA16 virus-like particles (VLPs) are promising vaccine candidates against HFMD and are currently under development. In this paper, the influence of insect cell line, incubation temperature, and serial passaging effect and stability of budded virus (BV) stocks on EV71 and CVA16 VLP production was investigated. Enhanced EV71 and CVA16 VLP production was observed in Sf9 cells compared to High Five™ cells. Lowering the incubation temperature from the standard 27°C to 21°C increased the production of both VLPs in Sf9 cells. Serial passaging of CVA16 BV stocks in cell culture had a detrimental effect on the productivity of the structural proteins and the effect was observed with only 5 passages of BV stocks. A 2.7× higher production yield was achieved with EV71 compared to CVA16. High-resolution asymmetric flow field-flow fractionation couple with multi-angle light scattering (AF4-MALS) was used for the first time to characterize EV71 and CVA16 VLPs, displaying an average root mean square radius of 15±1nm and 15.3±5.8 nm respectively. This study highlights the need for different approaches in the design of production process to develop a bivalent EV71 and CVA16 vaccine.
Assuntos
Enterovirus Humano A/imunologia , Enterovirus/imunologia , Vacinas de Partículas Semelhantes a Vírus/biossíntese , Vacinas Virais/biossíntese , Vírion/genética , Animais , Anticorpos Antivirais/biossíntese , Baculoviridae/genética , Baculoviridae/metabolismo , Enterovirus/genética , Enterovirus Humano A/genética , Doença de Mão, Pé e Boca/imunologia , Doença de Mão, Pé e Boca/prevenção & controle , Doença de Mão, Pé e Boca/virologia , Humanos , Mimetismo Molecular , Coelhos , Células Sf9 , Spodoptera , Temperatura , Vacinas de Partículas Semelhantes a Vírus/genética , Vacinas de Partículas Semelhantes a Vírus/imunologia , Vacinas Virais/genética , Vacinas Virais/imunologia , Vírion/imunologiaRESUMO
Human interferon gamma (hIFNγ) is an important cytokine in the innate and adaptive immune system, produced commercially in Escherichia coli. Efficient expression of hIFNγ has been reported once for Pichia pastoris (Wang et al., 2014) - a proven heterologous expression system. This study investigated hIFNγ expression in P. pastoris replicating the previous study and expanding by using four different strains (X33: wild type; GS115: HIS-Mut+; KM71H: Arg+, Mut- and CBS7435: MutS) and three different vectors (pPICZαA, pPIC9 and pPpT4αS). In addition, the native sequence (NS) and two codon-optimised sequences (COS1 and COS2) for P. pastoris were used. Methanol induction yielded no expression/secretion of hIFNγ in X33, highest levels were recorded for CBS7435: MutS (â¼16 µg. L-1). mRNA copy number calculations acquired from RT-qPCR for GS115-pPIC9-COS1 proved low abundance of mRNA. A 10-fold increase in expression of hIFNγ was achieved by lowering the minimal free energy of the mRNA and 100-fold by MutS phenotypes, substantially lower than reported by Wang et al. (2014). We conclude that commercial production of low cost, eukaryotic recombinant hIFNγ is not an economically viable in P. pastoris. Further research is required to unravel the cause of low expression in P. pastoris to achieve economic viability.
Assuntos
Interferon gama/biossíntese , Pichia/metabolismo , Humanos , Interferon gama/genética , Pichia/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genéticaRESUMO
Cattle tick infestations remain an important burden for farmers in tropical area like in New Caledonia. With the development of acaricide resistance, tick vaccines should be an attractive alternative to control ticks but their efficacy needs to be improved. In this study three adjuvants were studied in an experimental tick vaccine with a Bm86 protein to assess their performance in terms of antibody productions and adverse reactions following vaccinations. The water-in-oil adjuvant ISA 61 VG led to higher antibody titers compared to a water-in-oil-in-water adjuvant ISA 201 VG and an aqueous polymeric adjuvant Montanide Gel 01. Vaccinations with these three adjuvants did not produce severe general reaction but an increase in skin thickness was observed especially with both oil-based emulsions. These results indicated that the water-in-oil adjuvant is the most interesting to use for this vaccine but local adverse reactions remain an issue.
Assuntos
Doenças dos Bovinos/prevenção & controle , Rhipicephalus/imunologia , Infestações por Carrapato/veterinária , Vacinas/imunologia , Animais , Formação de Anticorpos , Bovinos , Doenças dos Bovinos/imunologia , Glicoproteínas de Membrana , Nova Caledônia , Proteínas Recombinantes , Rhipicephalus/crescimento & desenvolvimento , Infestações por Carrapato/imunologia , Infestações por Carrapato/parasitologia , Infestações por Carrapato/prevenção & controleRESUMO
Virus-like particles (VLPs) are repetitive organizations of viral proteins assembled in an appropriate physicochemical environment. VLPs can stimulate both innate and adaptive immune responses, due to their particulate structure enabling uptake by antigen presenting cells. These characteristics have led to successful development of VLP-vaccine products, and will ensure their vast potential in years to come. Future success of VLP therapeutic products will be determined by advances in their bioengineering, and also by the development of tools to design for their stability, function and application. This review focuses on approaches for VLP assembly in controlled chemical environments in vivo and in vitro, and the application of computational tools for improved chemical sequence design, and fundamental understanding of assembly.
Assuntos
Bioengenharia , Biologia Computacional , Modelos Moleculares , VírionRESUMO
Virus-like particle (VLP) technology seeks to harness the optimally tuned immunostimulatory properties of natural viruses while omitting the infectious trait. VLPs that assemble from a single protein have been shown to be safe and highly efficacious in humans, and highly profitable. VLPs emerging from basic research possess varying levels of complexity and comprise single or multiple proteins, with or without a lipid membrane. Complex VLP assembly is traditionally orchestrated within cells using black-box approaches, which are appropriate when knowledge and control over assembly are limited. Recovery challenges including those of adherent and intracellular contaminants must then be addressed. Recent commercial VLPs variously incorporate steps that include VLP in vitro assembly to address these problems robustly, but at the expense of process complexity. Increasing research activity and translation opportunity necessitate bioengineering advances and new bioprocessing modalities for efficient and cost-effective production of VLPs. Emerging approaches are necessarily multi-scale and multi-disciplinary, encompassing diverse fields from computational design of molecules to new macro-scale purification materials. In this review, we highlight historical and emerging VLP vaccine approaches. We overview approaches that seek to specifically engineer a desirable immune response through modular VLP design, and those that seek to improve bioprocess efficiency through inhibition of intracellular assembly to allow optimal use of existing purification technologies prior to cell-free VLP assembly. Greater understanding of VLP assembly and increased interdisciplinary activity will see enormous progress in VLP technology over the coming decade, driven by clear translational opportunity.
Assuntos
Bioengenharia/métodos , Vacinas de Partículas Semelhantes a Vírus/imunologia , Vacinas de Partículas Semelhantes a Vírus/isolamento & purificação , História do Século XX , História do Século XXI , Vacinas de Partículas Semelhantes a Vírus/históriaRESUMO
Effective and low-cost vaccines are essential to control severe group A streptococcus (GAS) infections prevalent in low-income nations and the Australian aboriginal communities. Highly diverse and endemic circulating GAS strains mandate broad-coverage and customized vaccines. This study describes an approach to deliver cross-reactive antigens from endemic GAS strains using modular virus-like particle (VLP) and capsomere systems. The antigens studied were three heterologous N-terminal peptides (GAS1, GAS2, and GAS3) from the GAS surface M-protein that are specific to endemic strains in Australia Northern Territory Aboriginal communities. In vivo data presented here demonstrated salient characteristics of the modular delivery systems in the context of GAS vaccine design. First, the antigenic peptides, when delivered by unadjuvanted modular VLPs or adjuvanted capsomeres, induced high titers of peptide-specific IgG antibodies (over 1 × 10(4) ). Second, delivery by capsomere was superior to VLP for one of the peptides investigated (GAS3), demonstrating that the delivery system relative effectiveness was antigen-dependant. Third, significant cross-reactivity of GAS2-induced IgG with GAS1 was observed using either VLP or capsomere, showing the possibility of broad-coverage vaccine design using these delivery systems and cross-reactive antigens. Fourth, a formulation containing three pre-mixed modular VLPs, each at a low dose of 5 µg (corresponding to <600 ng of each GAS peptide), induced significant titers of IgGs specific to each peptide, demonstrating that a multivalent, broad-coverage VLP vaccine formulation was possible. In summary, the modular VLPs and capsomeres reported here demonstrate, with promising preliminary data, innovative ways to design GAS vaccines using VLP and capsomere delivery systems amenable to microbial synthesis, potentially adoptable by developing countries.
Assuntos
Antígenos de Bactérias/metabolismo , Proteínas da Membrana Bacteriana Externa/imunologia , Proteínas de Transporte/imunologia , Vacinas Estreptocócicas/imunologia , Streptococcus pyogenes/imunologia , Vacinas Sintéticas/imunologia , Vacinas Virossomais/imunologia , Virossomos/metabolismo , Animais , Anticorpos Antibacterianos/sangue , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Transporte/genética , Reações Cruzadas , Humanos , Imunoglobulina G/sangue , Camundongos , Havaiano Nativo ou Outro Ilhéu do Pacífico , Northern Territory/epidemiologia , Infecções Estreptocócicas/epidemiologia , Infecções Estreptocócicas/prevenção & controle , Vacinas Estreptocócicas/administração & dosagem , Vacinas Estreptocócicas/genética , Vacinas Estreptocócicas/isolamento & purificação , Streptococcus pyogenes/genética , Vacinas de Subunidades Antigênicas/administração & dosagem , Vacinas de Subunidades Antigênicas/genética , Vacinas de Subunidades Antigênicas/imunologia , Vacinas de Subunidades Antigênicas/isolamento & purificação , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/isolamento & purificação , Vacinas Virossomais/administração & dosagem , Vacinas Virossomais/genética , Vacinas Virossomais/isolamento & purificação , Virossomos/genéticaRESUMO
Virus-like particles (VLPs) are non-infectious and immunogenic virus-mimicking protein assemblies that are increasingly researched as vaccine candidates. Stability against aggregation is an important determinant dictating the viability of a pipeline VLP product, making multivariable stability data highly desirable especially in early product development stages. However, comprehensive formulation studies are challenging due to low sample availability early in developability assessment. This issue is exacerbated by industry-standard analytical techniques which are low-throughput and/or sample-consuming. This study presents a miniaturized high-throughput screening (MHTS) methodology for VLP formulation by integrating dynamic light scattering (DLS) and asymmetrical flow field-flow fractionation (AF4) in a formulation funnel analysis. Using only 2 µg of sample and 100 s per measurement, a DLS plate reader was deployed to effectively pre-screen a large experimental space, allowing a smaller set of superior formulation conditions to be interrogated at high-resolution with AF4. The stabilizing effects of polysorbate 20, sucrose, trehalose, mannitol and sorbitol were investigated. MHTS data showed that addition of 0.5% w/v polysorbate 20 together with either 40% w/v sucrose or 40% w/v sorbitol could stabilize VLPs at elevated temperatures up to 58 °C. AF4 data further confirmed that the formulation containing 40% w/v sorbitol and 0.5% w/v polysorbate 20 effectively protected VLPs during freeze-thawing and freeze-drying, increasing recoveries from these processes by 80 and 50 percentage points, respectively. The MHTS strategy presented here could be used to rapidly explore a large formulation development space using reduced amounts of sample, without sacrificing the analytical resolution needed for quality control. Such a method paves the way for rapid formulation development and could potentially hasten the commercialization of new VLP vaccines.
Assuntos
Ensaios de Triagem em Larga Escala , Vacinas de Partículas Semelhantes a Vírus/química , Vírion/química , Química Farmacêutica , Estabilidade de Medicamentos , Excipientes/química , Fracionamento por Campo e Fluxo , Liofilização , Luz , Polissorbatos/química , Espalhamento de Radiação , Sorbitol/química , Sacarose/químicaRESUMO
Munc18-1 and Syntaxin1 are essential proteins for SNARE-mediated neurotransmission. Munc18-1 participates in synaptic vesicle fusion via dual roles: as a docking/chaperone protein by binding closed Syntaxin1, and as a fusion protein that binds SNARE complexes in a Syntaxin1 N-peptide dependent manner. The two roles are associated with a closed-open Syntaxin1 conformational transition. Here, we show that Syntaxin N-peptide binding to Munc18-1 is not highly selective, suggesting that other parts of the SNARE complex are involved in binding to Munc18-1. We also find that Syntaxin1, with an N peptide and a physically anchored C terminus, binds to Munc18-1 and that this complex can participate in SNARE complex formation. We report a Munc18-1-N-peptide crystal structure that, together with other data, reveals how Munc18-1 might transit from a conformation that binds closed Syntaxin1 to one that may be compatible with binding open Syntaxin1 and SNARE complexes. Our results suggest the possibility that structural transitions occur in both Munc18-1 and Syntaxin1 during their binary interaction. We hypothesize that Munc18-1 domain 3a undergoes a conformational change that may allow coiled-coil interactions with SNARE complexes.
Assuntos
Modelos Moleculares , Complexos Multiproteicos/metabolismo , Proteínas Munc18/metabolismo , Conformação Proteica , Proteínas SNARE/metabolismo , Transmissão Sináptica/fisiologia , Sintaxina 1/metabolismo , Sequência de Aminoácidos , Dicroísmo Circular , Cristalografia , Dados de Sequência Molecular , Proteínas Munc18/química , Ligação Proteica , Alinhamento de SequênciaRESUMO
The caspase recruitment domain (CARD) is present in death-domain superfamily proteins involved in inflammation and apoptosis. BinCARD is named for its ability to interact with Bcl10 and inhibit downstream signalling. Human BinCARD is expressed as two isoforms that encode the same N-terminal CARD region but which differ considerably in their C-termini. Both isoforms are expressed in immune cells, although BinCARD-2 is much more highly expressed. Crystals of the CARD fold common to both had low symmetry (space group P1). Molecular replacement was unsuccessful in this low-symmetry space group and, as the construct contains no methionines, first one and then two residues were engineered to methionine for MAD phasing. The double-methionine variant was produced as a selenomethionine derivative, which was crystallized and the structure was solved using data measured at two wavelengths. The crystal structures of the native and selenomethionine double mutant were refined to high resolution (1.58 and 1.40â Å resolution, respectively), revealing the presence of a cis-peptide bond between Tyr39 and Pro40. Unexpectedly, the native crystal structure revealed that all three cysteines were oxidized. The mitochondrial localization of BinCARD-2 and the susceptibility of its CARD region to redox modification points to the intriguing possibility of a redox-regulatory role.
Assuntos
Proteínas Adaptadoras de Sinalização CARD/química , Proteínas/química , Proteínas/metabolismo , Proteínas Adaptadoras de Sinalização CARD/genética , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Cristalografia por Raios X , Cisteína/metabolismo , Células HeLa , Humanos , Mitocôndrias/metabolismo , Modelos Moleculares , Mutação , Oxirredução , Prolina/química , Conformação Proteica , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína , Proteínas/genética , SelenometioninaRESUMO
Modularization of a peptide antigen for presentation on a microbially synthesized murine polyomavirus (MuPyV) virus-like particle (VLP) offers a new alternative for rapid and low-cost vaccine delivery at a global scale. In this approach, heterologous modules containing peptide antigenic elements are fused to and displayed on the VLP carrier, allowing enhancement of peptide immunogenicity via ordered and densely repeated presentation of the modules. This study addresses two key engineering questions pertaining to this platform, exploring the effects of (i) pre-existing carrier-specific immunity on modular VLP vaccine effectiveness and (ii) increase in the antigenic element number per VLP on peptide-specific immune response. These effects were studied in a mouse model and with modular MuPyV VLPs presenting a group A streptococcus (GAS) peptide antigen, J8i. The data presented here demonstrate that immunization with a modular VLP could induce high levels of J8i-specific antibodies despite a strong pre-existing anti-carrier immune response. Doubling of the J8i antigenic element number per VLP did not enhance J8i immunogenicity at a constant peptide dose. However, the strategy, when used in conjunction with increased VLP dose, could effectively increase the peptide dose up to 10-fold, leading to a significantly higher J8i-specific antibody titer. This study further supports feasibility of the MuPyV modular VLP vaccine platform by showing that, in the absence of adjuvant, modularized GAS antigenic peptide at a dose as low as 150 ng was sufficient to raise a high level of peptide-specific IgGs indicative of bactericidal activity.
Assuntos
Antígenos/imunologia , Peptídeos/imunologia , Polyomavirus/imunologia , Vacinas de Partículas Semelhantes a Vírus , Animais , Portadores de Fármacos , Eletroforese em Gel de Poliacrilamida , Imunoglobulina G/sangue , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Vacinas de Partículas Semelhantes a Vírus/administração & dosagem , Vacinas de Partículas Semelhantes a Vírus/química , Vacinas de Partículas Semelhantes a Vírus/imunologiaRESUMO
Escherichia coli remains a traditional and widely used host organism for recombinant protein production. Its well-studied genome, availability of vectors and strains, cheap and relatively straight-forward cultivation methods paired with reported high protein yields are reasons why E. coli is often the first-choice host expression system for recombinant protein production. The chapter enclosed here details protocols and design strategies in strain selection and methods on how to parallelize expression conditions to optimize for soluble target protein expression in E. coli. The methods described have been validated in a protein production research facility.
Assuntos
Escherichia coli , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Recombinantes/metabolismoRESUMO
Organophosphorus nerve agents represent a serious chemical threat due to their ease of production and scale of impact. The recent use of the nerve agent Novichok has re-emphasised the need for broad-spectrum medical countermeasures (MCMs) to these agents. However, current MCMs are limited. Plasma derived human butyrylcholinesterase (huBChE) is a promising novel bioscavenger MCM strategy, but is prohibitively expensive to isolate from human plasma at scale. Efforts to produce recombinant huBChE (rBChE) in various protein expression platforms have failed to achieve key critical attributes of huBChE such as circulatory half-life. These proteins often lack critical features such as tetrameric structure and requisite post-translational modifications. This review evaluates previous attempts to generate rBChE and assesses recent advances in mammalian cell expression and protein engineering strategies that could be deployed to achieve the required half-life and yield for a viable rBChE MCM. This includes the addition of a proline-rich attachment domain, fusion proteins, post translational modifications, expression system selection and optimised downstream processes. Whilst challenges remain, a combinatorial application of these strategies demonstrates potential as a technically feasible approach to achieving a bioactive and cost effective bioscavenger MCM.
Assuntos
Contramedidas Médicas , Agentes Neurotóxicos , Intoxicação por Organofosfatos , Animais , Butirilcolinesterase/química , Humanos , Mamíferos/metabolismo , Intoxicação por Organofosfatos/tratamento farmacológico , Compostos Organofosforados , Proteínas Recombinantes/químicaRESUMO
The manufacturing scale implementation of membrane chromatography to purify monoclonal antibodies has gradually increased with the shift in industry focus toward flexible manufacturing and disposable technologies. Membrane chromatography are used to remove process-related impurities such as host cell proteins (HCPs) and DNA, leachates, and endotoxins, with improved productivity and process flexibility. However, application of membrane chromatography to separate product-related variants such as charge variants has not gained major traction due to low-binding capacity. The work reported here demonstrates that a holistic process development strategy to optimize static binding (pH and salt concentration) and dynamic process (membrane loading, flowrate, and gradient length) parameters can alleviate the capacity limitations. The study employed high throughput screening tools and scale-down membranes for intermediate and polishing purification of the model monoclonal antibody. An optimized process consisting of anion exchange and cation exchange membrane chromatography reduced the acidic variants present in Protein A eluate from 89.5% to 19.2% with 71% recovery of the target protein. The membrane chromatography process also cleared HCP to below limit of detection with 6- to 30-fold higher membrane loading, compared to earlier reported values. The results confirm that membrane chromatography is effective in separating closely related product variants when supported by a well-defined process development strategy.
Assuntos
Anticorpos Monoclonais , Cloreto de Sódio , Anticorpos Monoclonais/química , Cromatografia por Troca Iônica/métodos , Ânions , CátionsRESUMO
There is an unmet need for safe and effective severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines that are stable and can be cost-effectively produced at large scale. Here, a biopolymer particle (BP) vaccine technology that can be quickly adapted to new and emerging variants of SARS-CoV-2 is used. Coronavirus antigen-coated BPs are described as vaccines against SARS-CoV-2. The spike protein subunit S1 or epitopes from S and M proteins (SM) plus/minus the nucleocapsid protein (N) are selected as antigens to either coat BPs during assembly inside engineered Escherichia coli or BPs are engineered to specifically ligate glycosylated spike protein (S1-ICC) produced by using baculovirus expression in insect cell culture (ICC). BP vaccines are safe and immunogenic in mice. BP vaccines, SM-BP-N and S1-ICC-BP induced protective immunity in the hamster SARS-CoV-2 infection model as shown by reduction of virus titers up to viral clearance in lungs post infection. The BP platform offers the possibility for rapid design and cost-effective large-scale manufacture of ambient temperature stable and globally available vaccines to combat the coronavirus disease 2019 (COVID-19) pandemic.
Assuntos
Vacinas contra COVID-19 , COVID-19 , Animais , Anticorpos Antivirais , Cricetinae , Humanos , Camundongos , Polímeros , SARS-CoV-2 , TemperaturaRESUMO
Beta defensins are antimicrobial peptides (AMPs) with a broad spectrum antimicrobial behavior against pathogens while having minimal tendency to incur pathogen resistance. Human ß-defensin 28 (hBD28) is a strongly cationic AMP and hence hypothesized to be highly effective in permeabilizing negatively-charged pathogen membranes. However, the scarcity of hBD28 in vivo has impeded detailed structure and antimicrobial studies of hBD28. Chemical synthesis of hBD28 rendered extremely poor yields due to inefficient cysteine oxidation. In this study, a rapid and scalable production route to produce bioactive hBD28 in Escherichia coli (E. coli) is reported. The design of a dual fusion tag expression construct was pivotal in enhancing soluble expression and easing purification of hBD28. The final hBD28 (purity >95%) displayed significant antimicrobial activity against E. coli K12 and showed dose-dependent killing kinetics. Circular dichroism spectroscopy confirmed the presence of both ß-sheet and α-helix conformations in the secondary structure of hBD28.