Augmenting glycosylation-directed folding pathways enhances the fidelity of HIV Env immunogen production in plants.

Heterologous glycoprotein production relies on host glycosylation-dependent folding. When the biosynthetic machinery differs from the usual expression host, there is scope to remodel the assembly pathway to enhance glycoprotein production. Here we explore the integration of chaperone coexpression with glyco-engineering to improve the production of a model HIV-1 envelope antigen. Calreticulin was coexpressed to support protein folding together with Leishmania major STT3D oligosaccharyltransferase, to improve glycan occupancy, RNA interference to suppress the formation of truncated glycans, and Nicotiana benthamiana plants lacking α1,3-fucosyltransferase and ß1,2-xylosyltransferase was used as an expression host to prevent plant-specific complex N-glycans forming. This approach reduced the formation of undesired aggregates, which predominated in the absence of glyco-engineering. The resulting antigen also exhibited increased glycan occupancy, albeit to a slightly lower level than the equivalent mammalian cell-produced protein. The antigen was decorated almost exclusively with oligomannose glycans, which were less processed compared with the mammalian protein. Immunized rabbits developed comparable immune responses to the plant-produced and mammalian cell-derived antigens, including the induction of autologous neutralizing antibodies when the proteins were used to boost DNA and modified vaccinia Ankara virus-vectored vaccines. This study demonstrates that engineering glycosylation-directed folding offers a promising route to enhance the production of complex viral glycoproteins in plants.

Characterization of a Novel Chimeric Theileria parva p67 Antigen Which Incorporates into Virus-like Particles and Is Highly Immunogenic in Mice.

The current method to protect cattle against East Coast Fever (ECF) involves the use of live Theileria parva sporozoites. Although this provides immunity, using live parasites has many disadvantages, such as contributing to the spread of ECF. Subunit vaccines based on the sporozoite surface protein p67 have been investigated as a replacement for the current method. In this study, two DNA vaccines expressing recombinant forms of p67 designed to display on retrovirus-like particles were constructed with the aim of improving immunogenicity. The native leader sequence was replaced with the human tissue plasminogen activator leader in both vaccines. The full-length p67 gene was included in the first DNA vaccine (p67); in the second, the transmembrane domain and cytoplasmic tail were replaced with those of an influenza A virus hemagglutinin 5 (p67HA). Immunofluorescent staining of fixed and live transfected mammalian cells showed that both p67 and p67HA were successfully expressed, and p67HA localised on the cell surface. Furthermore, p67HA was displayed on the surface of both bovine leukaemia virus (BLV) Gag and HIV-1 Gag virus-like particles (VLPs) made in the same cells. Mice vaccinated with DNA vaccines expressing p67 and p67HA alone, or p67HA with BLV or HIV-1 Gag, developed high titres of p67 and BLV Gag-binding antibodies. Here we show that it is possible to integrate a form of p67 containing all known antigenic domains into VLPs. This p67HA-VLP combination has the potential to be incorporated into a vaccine against ECF, as a DNA vaccine or as other vaccine platforms.

Site-Specific Glycosylation of Recombinant Viral Glycoproteins Produced in Nicotiana benthamiana.

There is an urgent need to establish large scale biopharmaceutical manufacturing capacity in Africa where the infrastructure for biologics production is severely limited. Molecular farming, whereby pharmaceuticals are produced in plants, offers a cheaper alternative to mainstream expression platforms, and is amenable to rapid large-scale production. However, there are several differences along the plant protein secretory pathway compared to mammalian systems, which constrain the production of complex pharmaceuticals. Viral envelope glycoproteins are important targets for immunization, yet in some cases they accumulate poorly in plants and may not be properly processed. Whilst the co-expression of human chaperones and furin proteases has shown promise, it is presently unclear how plant-specific differences in glycosylation impact the production of these proteins. In many cases it may be necessary to reproduce features of their native glycosylation to produce immunologically relevant vaccines, given that glycosylation is central to the folding and immunogenicity of these antigens. Building on previous work, we transiently expressed model glycoproteins from HIV and Marburg virus in Nicotiana benthamiana and mammalian cells. The proteins were purified and their site-specific glycosylation was determined by mass-spectrometry. Both glycoproteins yielded increased amounts of protein aggregates when produced in plants compared to the equivalent mammalian cell-derived proteins. The glycosylation profiles of the plant-produced glycoproteins were distinct from the mammalian cell produced proteins: they displayed lower levels of glycan occupancy, reduced complex glycans and large amounts of paucimannosidic structures. The elucidation of the site-specific glycosylation of viral glycoproteins produced in N. benthamiana is an important step toward producing heterologous viral glycoproteins in plants with authentic human-like glycosylation.

Co-expression of human calreticulin significantly improves the production of HIV gp140 and other viral glycoproteins in plants.

Plant molecular farming (PMF) is rapidly gaining traction as a viable alternative to the currently accepted paradigm of producing biologics. While the platform is potentially cheaper and more scalable than conventional manufacturing systems, expression yields and appropriate post-translational modifications along the plant secretory pathway remain a challenge for certain proteins. Viral fusion glycoproteins in particular are often expressed at low yields in plants and, in some cases, may not be appropriately processed. Recently, however, transiently or stably engineering the host plant has shown promise as a strategy for producing heterologous proteins with more complex maturation requirements. In this study we investigated the co-expression of a suite of human chaperones to improve the production of a human immunodeficiency virus (HIV) type 1 soluble gp140 vaccine candidate in Nicotiana benthamiana plants. The co-expression of calreticulin (CRT) resulted in a dramatic increase in Env expression and ameliorated the endoplasmic reticulum (ER) stress response - as evidenced by lower transcript abundance of representative stress-responsive genes. The co-expression of CRT similarly improved accumulation of glycoproteins from Epstein-Barr virus (EBV), Rift Valley fever virus (RVFV) and chikungunya virus (CHIKV), suggesting that the endogenous chaperone machinery may impose a bottleneck for their production. We subsequently successfully combined the co-expression of human CRT with the transient expression of human furin, to enable the production of an appropriately cleaved HIV gp140 antigen. These transient plant host engineering strategies are a promising approach for the production of high yields of appropriately processed and cleaved viral glycoproteins.

Production and Immunogenicity of Soluble Plant-Produced HIV-1 Subtype C Envelope gp140 Immunogens.

The development of effective vaccines is urgently needed to curb the spread of human immunodeficiency virus type 1 (HIV-1). A major focal point of current HIV vaccine research is the production of soluble envelope (Env) glycoproteins which reproduce the structure of the native gp160 trimer. These antigens are produced in mammalian cells, which requires a sophisticated infrastructure for manufacture that is mostly absent in developing countries. The production of recombinant proteins in plants is an attractive alternative for the potentially cheap and scalable production of vaccine antigens, especially for developing countries. In this study, we developed a transient expression system in Nicotiana benthamiana for the production of soluble HIV Env gp140 antigens based on two rationally selected virus isolates (CAP256 SU and Du151). The scalability of the platform was demonstrated and both affinity and size exclusion chromatography (SEC) were explored for recovery of the recombinant antigens. Rabbits immunized with lectin affinity-purified antigens developed high titres of binding antibodies, including against the V1V2 loop region, and neutralizing antibodies against Tier 1 viruses. The removal of aggregated Env species by gel filtration resulted in the elicitation of superior binding and neutralizing antibodies. Furthermore, a heterologous prime-boost regimen employing a recombinant modified vaccinia Ankara (rMVA) vaccine, followed by boosts with the SEC-purified protein, significantly improved the immunogenicity. To our knowledge, this is the first study to assess the immunogenicity of a near-full length plant-derived Env vaccine immunogen.

The Axonal Membrane Protein PRG2 Inhibits PTEN and Directs Growth to Branches.

In developing neurons, phosphoinositide 3-kinases (PI3Ks) control axon growth and branching by positively regulating PI3K/PI(3,4,5)P3, but how neurons are able to generate sufficient PI(3,4,5)P3 in the presence of high levels of the antagonizing phosphatase PTEN is difficult to reconcile. We find that normal axon morphogenesis involves homeostasis of elongation and branch growth controlled by accumulation of PI(3,4,5)P3 through PTEN inhibition. We identify a plasma membrane-localized protein-protein interaction of PTEN with plasticity-related gene 2 (PRG2). PRG2 stabilizes membrane PI(3,4,5)P3 by inhibiting PTEN and localizes in nanoclusters along axon membranes when neurons initiate their complex branching behavior. We demonstrate that PRG2 is both sufficient and necessary to account for the ability of neurons to generate axon filopodia and branches in dependence on PI3K/PI(3,4,5)P3 and PTEN. Our data indicate that PRG2 is part of a neuronal growth program that induces collateral branch growth in axons by conferring local inhibition of PTEN.

Engineering FKBP-Based Destabilizing Domains to Build Sophisticated Protein Regulation Systems.

Targeting protein stability with small molecules has emerged as an effective tool to control protein abundance in a fast, scalable and reversible manner. The technique involves tagging a protein of interest (POI) with a destabilizing domain (DD) specifically controlled by a small molecule. The successful construction of such fusion proteins may, however, be limited by functional interference of the DD epitope with electrostatic interactions required for full biological function of proteins. Another drawback of this approach is the remaining endogenous protein. Here, we combined the Cre-LoxP system with an advanced DD and generated a protein regulation system in which the loss of an endogenous protein, in our case the tumor suppressor PTEN, can be coupled directly with a conditionally fine-tunable DD-PTEN. This new system will consolidate and extend the use of DD-technology to control protein function precisely in living cells and animal models.

Variomics screen identifies the re-entrant loop of the calcium-activated chloride channel ANO1 that facilitates channel activation.

The calcium-activated chloride channel ANO1 regulates multiple physiological processes. However, little is known about the mechanism of channel gating and regulation of ANO1 activity. Using a high-throughput, random mutagenesis-based variomics screen, we generated and functionally characterized ∼6000 ANO1 mutants and identified novel mutations that affected channel activity, intracellular trafficking, or localization of ANO1. Mutations such as S741T increased ANO1 calcium sensitivity and rendered ANO1 calcium gating voltage-independent, demonstrating a critical role of the re-entrant loop in coupling calcium and voltage sensitivity of ANO1 and hence in regulating ANO1 activation. Our data present the first unbiased and comprehensive study of the structure-function relationship of ANO1. The novel ANO1 mutants reported have diverse functional characteristics, providing new tools to study ANO1 function in biological systems, paving the path for a better understanding of the function of ANO1 and its role in health and diseases.

Phosphorylation of the actin binding protein Drebrin at S647 is regulated by neuronal activity and PTEN.

Defects in actin dynamics affect activity-dependent modulation of synaptic transmission and neuronal plasticity, and can cause cognitive impairment. A salient candidate actin-binding protein linking synaptic dysfunction to cognitive deficits is Drebrin (DBN). However, the specific mode of how DBN is regulated at the central synapse is largely unknown. In this study we identify and characterize the interaction of the PTEN tumor suppressor with DBN. Our results demonstrate that PTEN binds DBN and that this interaction results in the dephosphorylation of a site present in the DBN C-terminus--serine 647. PTEN and pS647-DBN segregate into distinct and complimentary compartments in neurons, supporting the idea that PTEN negatively regulates DBN phosphorylation at this site. We further demonstrate that neuronal activity increases phosphorylation of DBN at S647 in hippocampal neurons in vitro and in ex vivo hippocampus slices exhibiting seizure activity, potentially by inducing rapid dissociation of the PTEN:DBN complex. Our results identify a novel mechanism by which PTEN is required to maintain DBN phosphorylation at dynamic range and signifies an unusual regulation of an actin-binding protein linked to cognitive decline and degenerative conditions at the CNS synapse.

MyosinV controls PTEN function and neuronal cell size.

The tumour suppressor PTEN can inhibit cell proliferation and migration as well as control cell growth, in different cell types. PTEN functions predominately as a lipid phosphatase, converting PtdIns(3,4,5)P(3) to PtdIns(4,5)P(2), thereby antagonizing PI(3)K (phosphoinositide 3-kinase) and its established downstream effector pathways. However, much is unclear concerning the mechanisms that regulate PTEN movement to the cell membrane, which is necessary for its activity towards PtdIns(3,4,5)P(3) (Refs 3, 4, 5). Here we show a requirement for functional motor proteins in the control of PI3K signalling, involving a previously unknown association between PTEN and myosinV. FRET (Förster resonance energy transfer) measurements revealed that PTEN interacts directly with myosinV, which is dependent on PTEN phosphorylation mediated by CK2 and/or GSK3. Inactivation of myosinV-transport function in neurons increased cell size, which, in line with known attributes of PTEN-loss, required PI(3)K and mTor. Our data demonstrate a myosin-based transport mechanism that regulates PTEN function, providing new insights into the signalling networks regulating cell growth.

Function of PTEN during the formation and maintenance of neuronal circuits in the brain.

PTEN (phosphatase and tensin homologue deleted on chromosome 10) is a tumor suppressor that can inhibit proliferation and migration and controls apoptosis in a number of cell types, mainly through inhibition of the phosphoinositide 3-kinase (PI3K) signaling pathway. Patients carrying inactivating mutations of PTEN show a prevalence to develop tumors that can coincide with neurological defects such as mental retardation, ataxia and seizures. A number of in vitro and in vivo studies were instrumental in uncovering a direct correlation between deregulated PI3K/PTEN signaling and changes in neuronal morphogenesis, which is likely to have profound bearings upon the pathogenesis of neurological symptoms. This review outlines recent work on the function of PTEN during vertebrate brain development and the current understanding of the signaling pathways downstream of PTEN that control neuronal connectivity in the brain.