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Highlights • AQ1 diatom frustules were advantageously adopted for lysozyme purification from chicken egg white. • The surface area and cation exchange capacity of AQ1 frustules were about 400 m2/g and 140 μmol/mL. • Through batch purification, the lysozyme recovery was 80–89% with a purity of 90–98% by AQ1 frustules. • In flow-through mode, the lysozyme purity could be notably increased to 99%. Abstract A nontoxic chromatographic matrix, with low cost and high adsorption capacity, is always a major goal for therapeutic protein purification. In this study, the frustules from two cultured diatoms, Nitzschia bilobata (AQ1) and Psammodictyon panduriforme (NP), were investigated as cation exchange materials for lysozyme purification from chicken egg white. The surface area and cation exchange capacity of frustules were about 400 m2/g and 140 μmol/mL for AQ1, 390 m2/g and 130 µmol/mL for NP. The optimal pH was 9 for adsorption. Through batch purification, the lysozyme recovery was 86% with a purity of 95% by AQ1 frustules, which was higher than that by NP frustules (82% with a purity of 90%). In the flow-through system, the purity using AQ1 frustules notably increased to 99%, higher than the result of 91% using NP frustules. Diatom frustules from AQ1 are more effective and could be an alternative chromatographic matrix for lysozyme purification. [ABSTRACT FROM AUTHOR]

Abstract: Virus-like particles (VLPs) are a highly effective type of subunit vaccines because they mimic the viral capsid without the presence of infectious viral genetic material. In this study, hemagglutinin (HA) alone and in combination with matrix 1 (M1) were highly expressed in Hi-5 insect cells for the production of one-component (containing HA) H-VLP and two-component (containing HA and M1) influenza MH-VLP. The H-VLP or MH-VLP expressed in culture supernatant was purified in fractions with a strong hemagglutination response. Transmission electron microscopy analysis confirmed that the morphology of both H-VLP and MH-VLP was similar to that of authentic influenza virions. After 4 weeks of vaccination, hemagglutination inhibition (HAI) titers induced by either one dose (2μg of HA) or two doses (a dosage of 0.5 or 0.2μg of HA) of both types of VLPs in chickens were all more than 97, well above 16, the predictive protective HAI titer in chickens. These results confirm the effectiveness of insect cell-derived H-VLP and MH-VLP in avian influenza vaccine design. Most importantly, VLPs comprising HA alone provide a simple and fast platform for avian influenza vaccine manufacturing. [Copyright &y& Elsevier]

This study describes an alternative approach to produce rVP2H protein using insect larvae of the cabbage looper Trichoplusia ni as hosts for the expression of the protein. The chimeric rVP2H protein, having an extra six histidine residues at the C-terminus of the VP2, a structural protein of infectious bursal disease virus (IBDV), is a vaccine candidate for the prevention of infectious bursal disease. The chimeric rVP2H protein was expressed in insect larvae in form of virus-like particles, in which they maintain their native immunogenic properties. The expression level of rVP2H protein in T. ni larvae was estimated to be approximately 0.4 mg/g of larvae or 0.2 mg/larvae. The rVP2H particles have a uniform morphology of dodecahedral structure with a size of 23 nm in diameter, and the particles could be affinity-purified in one step with immobilized metal-ion affinity chromatography (IMAC) from the larvae homogenate. The recovery of rVP2H protein was approximately 55% following IMAC and the protein was obtained with a purity of around 90%. An additional purification step of ammonium sulphate precipitation was added to speed up the process of microfiltration and ultrafiltration of the homogenate prior to IMAC. This step enhanced the final purity of rVP2H protein to 99%, demonstrating that the purification protocol developed herein was a powerful strategy for obtaining highly pure rVP2H protein from insect larvae. The immunogenicity and protective properties of the larvae-derived rVP2H protein were evaluated using a chicken protection assay. When larvae-derived rVP2H protein was intramuscularly injected into specific-pathogen-free chickens (20 μg/bird), high titres of virus-neutralizing antibodies were induced and the chickens were protected from the infection of a very virulent strain of IBDV isolated locally. [Copyright &y& Elsevier]

Highlights: [•] VP4 protein of infectious bursal disease virus (IBDV) self-cleavages at a VP4 cleavage site in Escherichia coli and forms tubules thereafter. [•] Deletion of 28 amino acids at the C-terminus of VP4 resulted in both VP4 monomers and dimers instead of tubule formation. [•] The endopeptidase activity of these VP4 monomers and dimers was about 12.5 times higher than that of VP4 tubules. [•] Formation of VP4 tubules inhibits VP4 protease activity. [ABSTRACT FROM AUTHOR]

Abstract: The frustule of diatoms, through appropriate chemical modification, can be developed for a high adsorption level of recombinant proteins and viral nanoparticles. Field emission scanning electron microscopy (FE-SEM) analysis of clean frustules revealed a 3D loculate areolae structure (valvar phase porous pattern of the siliceous cell wall). Isocyanatopropyl triethoxysilane (IPS) and iminodiacetic acid (IDA) were used to immobilize Cu2+ ions (an average Cu2+ adsorption capacity about 190μmol of Cu2+/ml of the Cu2+-coupled biosilica reached). FE-SEM, energy dispersion X-ray spectroscopy (EDS) and Fourier transform infrared (FT-IR) were used to confirm the chemical modification of the Cu2+-coupled biosilica. Protein adsorption was confirmed with the detection of a recombinant (His)6-tagged green fluorescent protein binding using fluorescent microscopy. Infectious bursal disease virus VP2-441 subviral particles (SVPs) were found to bind to the Cu2+-coupled biosilica (approximately 3×10−9 mol of VP2-441 SVPs/ml of modified frustules), a level higher than the previously obtained 9×10−10 mol/ml for SVP binding using a commercial Ni–NTA resin. These give diatom frustules the potential to be developed into a material useful in viral nanoparticle purification systems or as a biosensor for the detection of viruses. [Copyright &y& Elsevier]

Abstract: VP2 is the primary host-protective immunogen of infectious bursal disease virus (IBDV), the agent that causes the highly contiguous infectious bursal disease (IBD). Previous studies have shown that a C-terminal his-tagged 452 amino acid residue VP2 precursor variant (VP2-452H) can form an immunogenic subviral particle (SVP). A set of his-tagged N- and C-terminal VP2-452 deleting mutants (designated as N5-452H, N10-452H, N20-452H, N40-452H, VP2-441H, VP2-437H, VP2-411H and VP2-399H) was expressed in insect cells to discover the role of both N- and C-termini on the assembly of SVP and to develop an efficient SVP-based vaccine. Among these mutants, the expression level of N5-452H was the highest. Results of ultracentrifugation and electron microscopy also indicated that mutants of N-terminal deletion N10-452H, N20-452H and N40-452H or C-terminal deletion VP2-411H and VP2-399H lost the capability to self-assemble SVP. The other mutants, N5-452H, VP2-441H and VP2-437H, formed SVP. Additionally, SVP formed by N5-452H could not only be single-step purified by immobilized metal-ion affinity chromatography (IMAC), but it could also induce a high titer of neutralizing activity to protect chicks from the infection of IBDV at a low dosage (0.2μg), suggesting that SVP formed by N5-452H can be an alternative vaccine candidate for the prevention of IBD. [Copyright &y& Elsevier]

Abstract: Infectious bursal disease virus (IBDV) causes a contagious immunosuppressive disease in chickens. The aim of the present study is to develop an enzyme-linked immunosorbent assay (ELISA) using the expressed VP2 or VP3 protein of IBDV as the coating antigen for detecting antibodies to IBDV. Experimental results were compared with virus neutralization assay and a commercial-available ELISA. These assays were used to examine the sera from farm chickens and chickens vaccinated experimentally. The VP3-based ELISA had a higher correlation coefficient (R 2) of 0.812 with a commercial ELISA kit at a serum dilution of 1:500 than that of VP2-based ELISA (R 2) of 0.671. The relative sensitivity between virus neutralization and VP2-ELISA and VP3-ELISA was 96% (251/262) and 100% (262/262), respectively, and that between virus neutralization and a commercial ELISA was 99% (257/261). Additionally, compared with virus neutralization assay, the reference technique for diagnosing IBDV, VP3-based ELISA had an agreement value of 99%, superior to that of VP2-based ELISA (95%) or the commercial kit (89%). These results revealed that the capability of either VP2-ELISA or VP3-ELISA in detecting the field chicken sera was comparable to the commercial one, which is generally used to replace the virus neutralization assay. However, the preparation of VP3 is derived from an Escherichia coli expression system with a high yield and purification efficiency by Ni2+-NTA gels, which is more favorable to the insect cell-derived particles formed by VP2. Therefore, VP3-ELISA could be developed as an efficient and low cost diagnostic method for IBDV infection in field chickens. [Copyright &y& Elsevier]