Summary 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.

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.

A vaccine regimen that elicits broadly neutralizing antibodies (bNAbs) is a major goal in HIV-1 vaccine research. In this study, we assessed the immunogenicity of the CAP256 superinfecting viral envelope (CAP256 SU) protein delivered by modified vaccinia virus Ankara (MVA) and DNA vaccines in different prime-boost combinations followed by a soluble protein (P) boost. The envelope protein (Env) contained a flexible glycine linker and I559P mutation. Trimer-specific bNAbs PGT145, PG16, and CAP256 VRC26_08 efficiently bound to the membrane-bound CAP256 envelope expressed on the surface of cells transfected or infected with the DNA and MVA vaccines. The vaccines were tested in two different vaccination regimens in rabbits. Both regimens elicited autologous tier 2 neutralizing antibodies (NAbs) and high-titer binding antibodies to the matching CAP256 Env and CAP256 V1V2 loop scaffold. The immunogenicity of DNA and MVA vaccines expressing membrane-bound Env alone was compared to that of Env stabilized in a more native-like conformation on the surface of Gag virus-like particles (VLPs). The inclusion of Gag in the DNA and MVA vaccines resulted in earlier development of tier 2 NAbs for both vaccination regimens. In addition, a higher proportion of the rabbits primed with DNA and MVA vaccines that included Gag developed tier 2 NAbs than did those primed with vaccine expressing Env alone. Previously, these DNA and MVA vaccines expressing subtype C mosaic HIV-1 Gag were shown to elicit strong T cell responses in mice. Here we show that when the CAP256 SU envelope protein is included, these vaccines elicit autologous tier 2 NAbs. IMPORTANCE A vaccine is urgently needed to combat HIV-1, particularly in sub-Saharan Africa, which remains disproportionately affected by the AIDS pandemic and accounts for the majority of new infections and AIDS-related deaths. In this study, two different vaccination regimens were compared. Rabbits that received two DNA primes followed by two modified vaccinia virus Ankara (MVA) and two protein inoculations developed better immune responses than those that received two MVA and three protein inoculations. In addition, DNA and MVA vaccines that expressed mosaic Gag VLPs presenting a stabilized Env antigen elicited better responses than Env alone, which supports the inclusion of Gag VLPs in an HIV-1 vaccine.

The tumour suppressor PTEN can inhibit proliferation and migration as well as control cell growth in different cell types1. PTEN functions predominately as a lipid phsophatase, converting PI(3,4,5)P3 to PI(4,5)P2, thereby antagonizing PI3K (Phosphoinositide 3-kinase) and its established downstream effector pathways2. However, much is unclear concerning the mechanisms that regulate PTEN movement to the cell membrane necessary for PTEN’s activity towards PI(3,4,5)P33-5.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 measurements revealed that PTEN interacts directly with MyosinV, 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-loss6, 7 - required PI3K and mTor. Our data demonstrate a myosin-based transport mechanism regulating PTEN function, providing new insights into the signalling networks regulating cell growth.

SummaryIn 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.