RESUMEN

Bordetella pertussis is the bacterial causative agent of whooping cough, a serious respiratory illness. An extensive knowledge on its virulence regulation and metabolism is a key factor to ensure pertussis vaccine manufacturing process robustness. The aim of this study was to refine our comprehension of B. pertussis physiology during in vitro cultures in bioreactors. A longitudinal multi-omics analysis was carried out over 26 h small-scale cultures of B. pertussis. Cultures were performed in batch mode and under culture conditions intending to mimic industrial processes. Putative cysteine and proline starvations were, respectively, observed at the beginning of the exponential phase (from 4 to 8 h) and during the exponential phase (18 h 45 min). As revealed by multi-omics analyses, the proline starvation induced major molecular changes, including a transient metabolism with internal stock consumption. In the meantime, growth and specific total PT, PRN, and Fim2 antigen productions were negatively affected. Interestingly, the master virulence-regulating two-component system of B. pertussis (BvgASR) was not evidenced as the sole virulence regulator in this in vitro growth condition. Indeed, novel intermediate regulators were identified as putatively involved in the expression of some virulence-activated genes (vags). Such longitudinal multi-omics analysis applied to B. pertussis culture process emerges as a powerful tool for characterization and incremental optimization of vaccine antigen production.

RESUMEN

Hemorrhagic fever outbreaks are difficult to diagnose and control in part because of a lack of low-cost and easily accessible diagnostic structures in countries where etiologic agents are present. Furthermore, initial clinical symptoms are common and shared with other endemic diseases such as malaria or typhoid fever. Current molecular diagnostic methods such as polymerase chain reaction require trained personnel and laboratory infrastructure, hindering diagnostics at the point of need, particularly in outbreak settings. Therefore, rapid diagnostic tests such as lateral flow can be broadly deployed and are typically well-suited to rapidly diagnose hemorrhagic fever viruses, such as Ebola virus. Early detection and control of Ebola outbreaks require simple, easy-to-use assays that can detect very low amount of virus in blood. Here, we developed and characterized an immunoassay test based on immunochromatography coupled to silver amplification technology to detect the secreted glycoprotein of EBOV. The glycoprotein is among the first viral proteins to be detected in blood. This strategy aims at identifying infected patients early following onset of symptoms by detecting low amount of sGP protein in blood samples. The limit of detection achieved by this sGP-targeted kit is 2.2 x 104 genome copies/ml in plasma as assayed in a monkey analytical cohort. Clinical performance evaluation showed a specificity of 100% and a sensitivity of 85.7% when evaluated with plasma samples from healthy controls and patients infected with Zaire Ebola virus from Macenta, Guinea. This rapid and accurate diagnostic test could therefore be used in endemic countries for early detection of infected individuals in point of care settings. Moreover, it could also support efficient clinical triage in hospitals or clinical centers and thus reducing transmission rates to prevent and better manage future severe outbreaks.

Asunto(s)

Antígenos Virales/aislamiento & purificación , Ebolavirus/aislamiento & purificación , Fiebre Hemorrágica Ebola/diagnóstico , Inmunoensayo , Ebolavirus/inmunología , Humanos , Inmunoensayo/métodos , Inmunoensayo/normas , Sistemas de Atención de Punto , Reproducibilidad de los Resultados

RESUMEN

CTX-M15 is one of the most widespread, extended spectrum ß-lactamases, a major determinant of antibiotic resistance representing urgent public health threats, among enterobacterial strains infecting humans and animals. Here we describe the selection of binders to CTX-M15 from a combinatorial affibody library displayed on ribosomes. Upon three increasingly selective ribosome display iterations, selected variants were identified by next generation sequencing (NGS). Nine affibody variants with high relative abundance bearing QRP and QLH amino acid motifs at residues 9-11 were produced and characterized in terms of stability, affinity and specificity. All affibodies were correctly folded, with affinities ranging from 0.04 to 2 µM towards CTX-M15, and successfully recognized CTX-M15 in bacterial lysates, culture supernatants and on whole bacteria. It was further demonstrated that the binding of affibody molecules to CTX-M15 modulated the enzyme's kinetic parameters. This work provides an approach using ribosome display coupled to NGS for the rapid generation of protein ligands of interest in diagnostic and research applications.

Asunto(s)

Ribosomas/metabolismo , beta-Lactamasas/metabolismo , Escherichia coli/enzimología , Klebsiella pneumoniae/enzimología , beta-Lactamasas/aislamiento & purificación

RESUMEN

In the past decades protein nanoparticles have successfully been used for vaccine applications. Their particulate nature and dense repetitive subunit organization makes them perfect carriers for antigen surface display and confers high immunogenicity. Nanoparticles have emerged as excellent candidates for vectorization of biological and immunostimulating molecules. Nanoparticles and biomolecular nanostructures such as ferritins or virus like particles have been used as diagnostic and therapeutic delivery systems, in vaccine development, as nanoreactors, etc. Recently, a new class of bacterial protein compartment has been discovered referred to as encapsulin nanocompartment. These compartments have been used for targeted diagnostics, as therapeutic delivery systems and as nanoreactors. Their biological origin makes them conveniently biocompatible and allows genetic functionalization. The aim of our study was to implement encapsulin nanocompartements for simultaneous epitope surface display and heterologous protein loading for rational vaccine design. For this proof-of-concept-study, we produced Thermotoga maritima encapsulin nanoparticles in E. coli. We demonstrated the ability of simultaneous display in our system by inserting Matrix protein 2 ectodomain (M2e) of influenza A virus at the nanoparticle surface and by packaging of a fluorescent reporter protein (GFP) into the internal cavity. Characterization of the nanoparticles by electronic microscopy confirmed homogenously shaped particles of 24 nm diameter in average. The results further show that engineering of the particle surface improved the loading capacity of the heterologous reporter protein suggesting that surface display may induce a critical elastic deformation resulting in improved stiffness. In Balb/c mice, nanoparticle immunization elicited antibody responses against both the surface epitope and the loaded cargo protein. These results confirm the potential of encapsulin nanocompartments for customized vaccine design and antigen delivery.

Asunto(s)

Anticuerpos Antivirales/biosíntesis , Proteínas Bacterianas/genética , Nanopartículas/química , Plásmidos/inmunología , Vacunas de ADN/genética , Proteínas de la Matriz Viral/genética , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/inmunología , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/inmunología , Inmunización , Ratones , Ratones Endogámicos BALB C , Nanopartículas/administración & dosificación , Nanopartículas/ultraestructura , Tamaño de la Partícula , Plásmidos/administración & dosificación , Plásmidos/química , Thermotoga maritima/genética , Thermotoga maritima/metabolismo , Vacunas de ADN/administración & dosificación , Vacunas de ADN/inmunología , Proteínas de la Matriz Viral/inmunología

RESUMEN

Codon usage distribution has been soundly used by nature to fine tune protein biogenesis. Alteration of the mRNA structure or sequential scheduling of codons can profoundly affect translation, thus altering protein yield, functionality, solubility, and proper folding. Building on these observations, here, we present an evaluation of different recently designed algorithms of sequence adaptation based on Codon Adaptation Index (CAI) profiling. The first algorithm globally harmonizes synonymous codons in the original sequence in full respect to the heterologous expression host codon usage. The second recodes the sequence in accordance with the native sequence CAI profile. Our data, generated on three model proteins, highlights the importance to consider gene recoding as a parameter itself for recombinant protein expression improvement.

Asunto(s)

Codón/genética , Biología Computacional/métodos , Regulación de la Expresión Génica , Algoritmos , Secuencia de Bases , Biosíntesis de Proteínas , Solubilidad

RESUMEN

The increasing demand for recombinant vaccine antigens or immunotherapeutic molecules calls into question the universality of current protein expression systems. Vaccine production can require relatively low amounts of expressed materials, but represents an extremely diverse category consisting of different target antigens with marked structural differences. In contrast, monoclonal antibodies, by definition share key molecular characteristics and require a production system capable of very large outputs, which drives the quest for highly efficient and cost-effective systems. In discussing expression systems, the primary assumption is that a universal production platform for vaccines and immunotherapeutics will unlikely exist. This review provides an overview of the evolution of traditional expression systems, including mammalian cells, yeast and E.coli, but also alternative systems such as other bacteria than E. coli, transgenic animals, insect cells, plants and microalgae, Tetrahymena thermophila, Leishmania tarentolae, filamentous fungi, cell free systems, and the incorporation of non-natural amino acids.

Asunto(s)

Expresión Génica , Factores Inmunológicos/aislamiento & purificación , Factores Inmunológicos/metabolismo , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Vacunas/aislamiento & purificación , Vacunas/metabolismo , Biotecnología/métodos , Vectores Genéticos , Biología Molecular/métodos , Tecnología Farmacéutica/métodos

RESUMEN

Virus-like particles (VLPs) are promising molecular structures for the design and construction of novel vaccines, diagnostic tools, and gene therapy vectors. Size, oligomer assembly and repetitiveness of epitopes are optimal features to induce strong immune responses. Several VLP-based vaccines are currently licensed and commercialized, and many vaccine candidates are now under preclinical and clinical studies. In recent years, the development of genetically engineered recombinant VLPs has accelerated the need for new, improved downstream processes. In particular, a rapid low cost purification process has been identified as a remaining key challenge in manufacturing process development. In the present study we set up a size-exclusion chromatography-based, scalable purification protocol for the purification of a VLP-based influenza A vaccine produced in Escherichia coli. Recombinant VLPs derived from the RNA bacteriophage MS2 displaying an epitope from the ectodomain of Matrix 2 protein from influenza A virus were produced and purified. The 3 steps purification protocol uses a recently developed multimodal size-exclusion chromatography medium (Capto™ Core 700) in combination with detergent extraction and size-exclusion polishing to reach a 89% VLP purity with a 19% yield. The combination of this downstream strategy following production in E. coli would be suited for production of VLP-based veterinary vaccines targeting livestock and companion animals where large amounts of doses must be produced at an affordable price.

Asunto(s)

Cromatografía en Gel/métodos , Epítopos/inmunología , Virus de la Influenza A/inmunología , Vacunas contra la Influenza/aislamiento & purificación , Vacunas de Partículas Similares a Virus/aislamiento & purificación , Animales , Epítopos/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Virus de la Influenza A/genética , Vacunas contra la Influenza/genética , Levivirus/genética , Infecciones por Orthomyxoviridae/prevención & control , Infecciones por Orthomyxoviridae/veterinaria , Vacunas Sintéticas/genética , Vacunas Sintéticas/aislamiento & purificación , Vacunas de Partículas Similares a Virus/genética , Medicina Veterinaria/métodos , Proteínas de la Matriz Viral/genética , Proteínas de la Matriz Viral/inmunología

RESUMEN

The continuously improving sophistication of molecular engineering techniques gives access to novel classes of bio-therapeutics and new challenges for their production in full respect of the strengthening regulations. Among these biologic agents are DNA based vaccines or gene therapy products and to a lesser extent genetically engineered live vaccines or delivery vehicles. The use of antibiotic-based selection, frequently associated with genetic manipulation of microorganism is currently undergoing a profound metamorphosis with the implementation and diversification of alternative selection means. This short review will present examples of alternatives to antibiotic selection and their context of application to highlight their ineluctable invasion of the bio-therapeutic world.

RESUMEN

BACKGROUND: The increasing regulatory requirements to which biological agents are subjected will have a great impact in the field of industrial protein expression and production. There is an expectation that in a near future, there may be "zero tolerance" towards antibiotic-based selection and production systems. Besides the antibiotic itself, the antibiotic resistance gene is an important consideration. The complete absence of antibiotic-resistance gene being the only way to ensure that there is no propagation in the environment or transfer of resistance to pathogenic strains. RESULTS: In a first step, we have designed a series of vectors, containing a stabilization element allowing a complete elimination of antibiotics during fermentation. Vectors were further improved in order to include alternative selection means such as the well known poison/antidote stabilization system. Eventually we propose an elegant positive pressure of selection ensuring the elimination of the antibiotic-resistance gene through homologous recombination. In addition, we have shown that the presence of an antibiotic resistance gene can indirectly reduce the amount of expressed protein, since even in absence of selection pressure the gene would be transcribed and account for an additional stress for the host during the fermentation process. CONCLUSIONS: We propose a general strategy combining plasmid stabilization and antibiotic-free selection. The proposed host/vector system, completely devoid of antibiotic resistance gene at the end of construction, has the additional advantage of improving recombinant protein expression and/or plasmid recovery.

Asunto(s)

Antibacterianos/farmacología , Escherichia coli/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Escherichia coli/genética , Fermentación , Vectores Genéticos , Plásmidos/química , Plásmidos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo