Enveloped virus-like particles (VLPs) are increasingly used as vaccines and immunotherapeutics. Frequently, very time consuming density gradient centrifugation techniques are used for purification of VLPs. However, the progress towards optimized large-scale VLP production increased the demand for fast, cost efficient and scale able purification processes. We developed a chromatographic procedure for purification of HIV-1 gag VLPs produced in CHO cells. The clarified and filtered cell culture supernatant was directly processed on an anion-exchange monolith. The majority of host cell impurities passed through the column, whereas the VLPs were eluted by a linear or step salt gradient; the major fraction of DNA was eluted prior to VLPs and particles in the range of 100-200nm in diameter could be separated into two fractions. The earlier eluted fraction was enriched with extracellular particles associated to exosomes or microvesicles, whereas the late eluting fractions contained the majority of most pure HIV-1 gag VLPs. DNA content in the exosome-containing fraction could not be reduced by Benzonase treatment which indicated that the DNA was encapsulated. Many exosome markers were identified by proteomic analysis in this fraction. We present a laboratory method that could serve as a basis for rapid downstream processing of enveloped VLPs. Up to 2000 doses, each containing 1×10(9) particles, could be processed with a 1mL monolith within 47min. The method compared to density gradient centrifugation has a 220-fold improvement in productivity.

EMBO Press is an editorially independent publishing platform for the development of EMBO scientific publications.

The rapid quantification of enveloped virus-like particles (VLPs) requires orthogonal methods to obtain reliable results. Three methods-nanoparticle tracking analysis (NTA), size-exclusion HPLC (SE-HPLC) with UV detection, and detection with multi-angle light scattering (MALS)-for quantification of enveloped VLPs have been compared, and the lower and upper limits of detection and quantification have been evaluated. NTA directly counts the enveloped VLPs, and a particle number is obtained with a lower limit of detection (LLOD) of 1.7×107part/mL and lower limit of quantification (LLOQ) of 3.4×108part/mL. SE-HPLC with UV detection was calibrated with standards characterized by NTA, and a LLOD of 6.9×109part/mL and LLOQ of 2.1×1010part/mL were found. SE-HPLC with MALS does not require a pre-calibrated sample because with a spherical model based on the Rayleigh-Gans-Debye approximation, the particle concentration can be directly deduced from the scattered light. A LLOD of 4.8×108part/mL and LLOQ of 2.1×109part/mL were measured and substantially lower compared to the UV method. The absolute particle concentration measured by SE-HPLC-MALS is one order of magnitude lower compared to measurement by NTA, which is explained by the wide size distribution of an enveloped VLP suspension. The model used for evaluation of light scattering data assumes monodisperse, homogeneous, and spherical particles.

Human infections with a novel influenza A H7N9 subtype virus were reported in China recently. The virus caused severe disease with high mortality rates and it raised concerns over its pandemic potential. Here, we assessed in the mouse model protective efficacy of single immunisations with low vaccine doses of insect cell-derived H7 virus-like particles, consisting of hemagglutinin and matrix protein. Vaccinated mice were fully protected and survived a stringent lethal challenge (100 mLD50) with H7N9, even after a single, unadjuvanted, low vaccine dose (0.03 μg). Serum analysis revealed broad reactivity and hemagglutination inhibition activity across a panel of divergent H7 strains. Moreover, we detected significant levels of cross-reactivity to related group 2 hemagglutinins. These data demonstrate that virus-like particle vaccines have the potential to induce broadly protective immunity against the novel H7N9 virus and a variety of other H7 strains.

The plant-pathogenic fungusGlomerella cingulata(anamorphColletotrichum gloeosporoides) secretes high levels of an FAD-dependent glucose dehydrogenase (GDH) when grown on tomato juice-supplemented media. To elucidate its molecular and catalytic properties, GDH was produced in submerged culture. The highest volumetric activity was obtained in shaking flasks after 6 days of cultivation (3400 U l⁻¹, 4.2 % of total extracellular protein). GDH is a monomeric protein with an isoelectric point of 5.6. The molecular masses of the glycoforms ranged from 95 to 135 kDa, but after deglycosylation, a single 68 kDa band was obtained. The absorption spectrum is typical for an FAD-containing enzyme with maxima at 370 and 458 nm and the cofactor is non-covalently bound. The preferred substrates are glucose and xylose. Suitable electron acceptors are quinones, phenoxy radicals, 2,6-dichloroindophenol, ferricyanide and ferrocenium hexafluorophosphate. In contrast, oxygen turnover is very low. The GDH-encoding gene was cloned and phylogenetic analysis of the translated protein reveals its affiliation to the GMC family of oxidoreductases. The proposed function of this quinone and phenoxy radical reducing enzyme is to neutralize the action of plant laccase, phenoloxidase or peroxidase activities, which are increased in infected plants to evade fungal attack.

Purpose of work: A comparative analysis of new and established insect cell lines, in regard to process relevant parameters, provide data that can be exploited for designing more robust and effective protein production processes. The baculovirus-insect cell expression system has been efficiently used for the production of heterologous proteins. Three different insect cell lines Tnao38, High Five and Sf9 were compared in terms of virus susceptibility, baculovirus production and product yield of an intra-cellularly (YFP) and extra-cellularly (influenza A virus hemagglutinin)-expressed recombinant protein. The Tnao38 and High Five cell lines exhibited higher (tenfold) susceptibility to baculovirus infection than Sf9 cells, whereas Sf9 cells showed a higher (100-fold) capacity for production of infectious virus particles. Analysis of recombinant protein expression revealed considerably higher product yields in Tnao38 and High Five cells as compared to Sf9 cells, for both model proteins. Overall, the two Trichoplusia ni-derived cell lines, High Five and Tnao38, were significantly more efficient in terms of secreting proteins such as the glycoprotein hemagglutinin of influenza A virus.