Developing a novel hepatitis B core – based antigen presentation system

Plant-produced proteins of pharmaceutical interest are beginning to reach the market, and the advantages of transient plant expression systems are gaining increasing recognition. In parallel, the use of virus-like particles (VLPs) has become standard in vaccine design. The pEAQ-HT vector derived from the cowpea mosaic virus – based CPMV-HT expression system has been shown to allow the production of large amounts of recombinant proteins, including VLPs, in Nicotiana benthamiana. Moreover, previous work demonstrated that a tandem fusion of the core antigen (HBcAg) of hepatitis B virus (HBV) could direct the formation of core-like particles (CLPs) in plants. The work presented here demonstrated that the tandem core system is better suited for the plant-based production of CLPs presenting foreign antigens than SplitCore technology. It was shown that tandem core technology allows the plant-based production of CLPs which are suitable for the presentation of antigens either via chemical coupling or through antibody-antigen interactions. Of particular significance was the successful display of single domain antibody fragments of camelid origin (nanobodies, or VHH). The resulting “tandibody” particles, as they are named here, can bind to their cognate antigen to yield CLPs covered in the antigen of interest. Furthermore, it was shown that target antigens can be attached to CLPs via a fusion partner, raising the possibility of the development of a universal generic antigen-display platform. In addition to the transient expression work on HBcAg, lines of stably transformed N. benthamiana were created which constitutively expressed human gastric lipase (hGL), an enzyme of medical importance. In the future, this approach could be used to create transgenic plants constitutively expressing a universal tandibody, thus obviating the need for infiltration

LoCKAmp: lab-on-PCB technology for <3 minute virus genetic detection

The recent COVID-19 outbreak highlighted the need for lab-on-chip diagnostic technology fit for real-life deployment in the field. Existing bottlenecks in multistep analytical microsystem integration and upscalable, standardized fabrication techniques delayed the large-scale deployment of lab-on-chip solutions during the outbreak, throughout a global diagnostic test shortage. This study presents a technology that has the potential to address these issues by redeploying and repurposing the ubiquitous printed circuit board (PCB) technology and manufacturing infrastructure. We demonstrate the first commercially manufactured, miniaturised lab-on-PCB device for loop-mediated isothermal amplification (LAMP) genetic detection of SARS-CoV-2. The system incorporates a mass-manufactured, continuous-flow PCB chip with ultra-low cost fluorescent detection circuitry, rendering it the only continuous-flow μLAMP platform with off-the-shelf optical detection components. Ultrafast, SARS-CoV-2 RNA amplification in wastewater samples was demonstrated within 2 min analysis, at concentrations as low as 17 gc μL−1. We further demonstrate our device operation by detecting SARS-CoV-2 in 20 human nasopharyngeal swab samples, without the need for any RNA extraction or purification. This renders the presented miniaturised nucleic-acid amplification-based diagnostic test the fastest reported SARS-CoV-2 genetic detection platform, in a practical implementation suitable for deployment in the field. This technology can be readily extended to the detection of alternative pathogens or genetic targets for a very broad range of applications and matrices. LoCKAmp lab-on-PCB chips are currently mass-manufactured in a commercial, ISO-compliant PCB factory, at a small-scale production cost of £2.50 per chip. Thus, with this work, we demonstrate a high technology-readiness-level lab-on-chip-based genetic detection system, successfully benchmarked against standard analytical techniques both for wastewater and nasopharyngeal swab SARS-CoV-2 detection

Tandem fusion of hepatitis B core antigen allows assembly of virus-like particles in bacteria and plants with enhanced capacity to accommodate foreign proteins

The core protein of the hepatitis B virus, HBcAg, assembles into highly immunogenic viruslike particles (HBc VLPs) when expressed in a variety of heterologous systems. Specifically, the major insertion region (MIR) on the HBcAg protein allows the insertion of foreign sequences, which are then exposed on the tips of surface spike structures on the outside of the assembled particle. Here, we present a novel strategy which aids the display of whole proteins on the surface of HBc particles. This strategy, named tandem core, is based on the production of the HBcAg dimer as a single polypeptide chain by tandem fusion of two HBcAg open reading frames. This allows the insertion of large heterologous sequences in only one of the two MIRs in each spike, without compromising VLP formation. We present the use of tandem core technology in both plant and bacterial expression systems. The results show that tandem core particles can be produced with unmodified MIRs, or with one MIR in each tandem dimer modified to contain the entire sequence of GFP or of a camelid nanobody. Both inserted proteins are correctly folded and the nanobody fused to the surface of the tandem core particle (which we name tandibody) retains the ability to bind to its cognate antigen. This technology paves the way for the display of natively folded proteins on the surface of HBc particles either through direct fusion or through non-covalent attachment via a nanobody

Efficient Production of Chimeric Hepatitis B Virus-Like Particles Bearing an Epitope of Hepatitis E Virus Capsid by Transient Expression in Nicotiana benthamiana

The core antigen of hepatitis B virus (HBcAg) is capable of self-assembly into virus-like particles (VLPs) when expressed in a number of heterologous systems. Such VLPs are potential carriers of foreign antigenic sequences for vaccine design. In this study, we evaluated the production of chimeric HBcAg VLPs presenting a foreign epitope on their surface, the 551&ndash;607 amino acids (aa) immunological epitope of the ORF2 capsid protein of hepatitis E virus. A chimeric construct was made by the insertion of 56 aa into the immunodominant loop of the HBcAg. The sequences encoding the chimera were inserted into the pEAQ-HT vector and infiltrated into Nicotiana benthamiana leaves. The plant-expressed chimeric HBcHEV ORF2 551&ndash;607 protein was recognized by an anti-HBcAg mAb and anti-HEV IgG positive swine serum. Electron microscopy showed that plant-produced chimeric protein spontaneously assembled into &ldquo;knobbly&rdquo; ~34 nm diameter VLPs. This study shows that HBcAg is a promising carrier platform for the neutralizing epitopes of hepatitis E virus (HEV) and the chimeric HBcAg/HEV VLPs could be a candidate for a bivalent vaccine

The hexosamine pathway and coat complex II promote malignant adaptation to nutrient scarcity

International audienceThe glucose-requiring hexosamine biosynthetic pathway (HBP), which produces UDP-N-acetylglucosamine for glycosylation reactions, promotes lung adenocarcinoma (LUAD) progression. However, lung tumor cells often reside in low-nutrient microenvironments, and whether the HBP is involved in the adaptation of LUAD to nutrient stress is unknown. Here, we show that the HBP and the coat complex II (COPII) play a key role in cell survival during glucose shortage. HBP up-regulation withstood low glucose-induced production of proteins bearing truncated N-glycans, in the endoplasmic reticulum. This function for the HBP, alongside COPII up-regulation, rescued cell surface expression of a subset of glycoproteins. Those included the epidermal growth factor receptor (EGFR), allowing an EGFR-dependent cell survival under low glucose in anchorage-independent growth. Accordingly, high expression of the HBP rate-limiting enzyme GFAT1 was associated with wild-type EGFR activation in LUAD patient samples. Notably, HBP and COPII up-regulation distinguished LUAD from the lung squamous-cell carcinoma subtype, thus uncovering adaptive mechanisms of LUAD to their harsh microenvironment

τGFP expressed in plants forms VLPs.

<p>a) Predicted structure of the τGFP tandibody protein (Swiss-Prot model): green: core 1, yellow: core 2, pink: anti-GFP nanobody, red: linkers. b) Western blot of crude plant extracts. C: empty vector control, τGFP: tandem HBcAg construct with anti-GFP VHH in the core 2 MIR, μGFP: monomeric HBcAg containing anti-GFP VHH in the MIR. The 39 kDa band found in all plant extracts is non-specific. c) Electron micrograph of plant-produced τGFP particles purified by sucrose cushion. Scale bar 100 nm.</p

Cryo-EM analysis of plant-produced CoHe-GFPL VLPs.

<p>a) Particles were flash-frozen in vitreous ice, then subjected to cryo-electron microscopy. Class averages were obtained from 441 individual particles using EMAN software. The expanded view (lower right) is of an average of all images used. b) 3D reconstruction of the particles using icosahedral symmetry, superimposed on the He map as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120751#pone.0120751.g003" target="_blank">Fig. 3</a>. The CoHe-GFPL map is coloured red-to-blue from the centre of the volume towards its edge; the He map is shown in grey.</p