Immunoglobulin G (IgG)-based fusion proteins have been widely exploited as a potential vaccine delivery platform but in the absence of exogenous adjuvants, the lack of robust immunity remains an obstacle. Here, we report on a key modification that overcomes that obstacle. Thus, we constructed an IgG-Fc vaccine platform for dengue, termed D-PCF, which in addition to a dengue antigen incorporates the cholera toxin non-toxic B subunit (CTB) as a molecular adjuvant, with all three proteins expressed as a single polypeptide. Following expression in Nicotiana benthamiana plants, the D-PCF assembled as polymeric structures of similar size to human IgM, a process driven by the pentamerization of CTB. A marked improvement of functional properties in vitro and immunogenicity in vivo over a previous iteration of the Fc-fusion protein without CTB [1] was demonstrated. These include enhanced antigen presenting cell binding, internalization and activation, complement activation, epithelial cell interactions and ganglioside binding, as well as more efficient polymerization within the expression host. Following immunization of mice with D-PCF by a combination of systemic and mucosal (intranasal) routes, we observed robust systemic and mucosal immune responses, as well as systemic T cell responses, significantly higher than those induced by a related Fc-fusion protein but without CTB. The induced antibodies could bind to the domain III of the dengue virus envelope protein from all four dengue serotypes. Finally, we also demonstrated feasibility of aerosolization of D-PCF as a prerequisite for vaccine delivery by the respiratory route.

The combination of scaffolds with recombinant human epidermal growth factor (rhEGF) protein can enhance defective bone healing via synergistic activation to stimulate cellular growth, differentiation, and survival. We examined the biopotentials of an rhEGF-loaded absorbable collagen scaffold (ACS) using a mouse model of calvarial defects, in which the rhEGF was produced from a plant cell suspension culture system because of several systemic advantages. Here, we showed a successful and large-scale production of plant-cell-derived rhEGF protein (p-rhEGF) by introducing an expression vector that cloned with its cDNA under the control of rice α-amylase 3D promoter into rice calli (Oryza sativa L. cv. Dongjin). Implantation with p-rhEGF (5 μg)-loaded ACSs into critical-sized calvarial defects enhanced new bone formation and the expression of osteoblast-specific markers in the defected regions greater than implantation with ACSs alone did. The potency of p-rhEGF-induced bone healing was comparable with that of Escherichia coli-derived rhEGF protein. The exogenous addition of p-rhEGF increased the proliferation of human periodontal ligament cells and augmented the induction of interleukin 8, bone morphogenetic protein 2, and vascular endothelial growth factor in the cells. Collectively, this study demonstrates the successful and convenient production of p-rhEGF, as well as its potency to enhance ACS-mediated bone regeneration by activating cellular responses that are required for wound healing.

Stem cells are an important therapeutic source for recovery and regeneration, as their ability of self-renewal and differentiation offers an unlimited supply of highly specialized cells for therapeutic transplantation. Growth factors and serum are essential for maintaining the characteristics of stem cells in culture and for inducing differentiation. Because growth factors are produced mainly in bacterial (Escherichia coli) or animal cells, the use of such growth factors raises safety concerns that need to be resolved for the commercialization of stem cell therapeutics. To overcome this problem, studies on proteins produced in plants have been conducted. Here, we describe the functions of plant-derived fibroblast growth factor 2 (FGF2) and human serum albumin in the maintenance and differentiation of human-induced pluripotent stem cells (hiPSCs). Plant-derived FGF2 and human epidermal growth factor EGF were able to differentiate hiPSCs into neural stem cells (NSCs). These NSCs could differentiate into neuronal and glial cells. Our results imply that culturing stem cells in animal-free culture medium, which is composed of plant-derived proteins, would facilitate stem cell application research, for example, for cell therapy, by reducing contamination risk.

Porcine epidemic diarrhea virus (PEDV), a member of the Coronaviridae family has become increasingly probelmatic in the pig farming industry. Currently, there are no effective, globally applicable vaccines against PEDV. Here, we tested a recombinant PEDV vaccine candidate based on the expression of the core neutralising epitope (COE) of PEDV conjugated to polymeric immunoglobulin G scaffold (PIGS) in glycoengineeredNicotiana benthamianaplants. The biological activity of COE-PIGS was demonstrated by binding to C1q component of the complement system, as well as the surface of antigen-presenting cells (APCs)in vitro.The recombinant COE-PIGS induced humoral and cellular immune responses specific for PEDV after both systemic and mucosal vaccination. Altogether, the data indicated that PEDV antigen fusion to poly-Fc could be a promising vaccine platform against respiratory PEDV infection.

Numerous studies have demonstrated the advantages of plant cell suspension culture systems in producing bioactive recombinant human growth factors. This study investigated the biological activity of recombinant basic human fibroblast growth factor (rhFGF2) protein produced by a plant culture system to enhance new bone formation in a bone defect mouse model. The human FGF2 cDNA gene was cloned into a plant expression vector driven by the rice α-amylase 3D promoter. The vector was introduced into rice calli (Oryza sativa L. cv. Dongjin), and the clone with the highest expression of rhFGF2 was selected. Maximum accumulation of rhFGF2 protein (approximately 28 mg/l) was reached at 13 day post-incubation. Male C57BL/6 mice underwent calvarial defect surgery and the defects were loaded with absorbable collagen sponge (ACS) only (ACS group) or ACS impregnated with 5 μg of plant-derived rhFGF2 (p-FGF2) protein or E. coli-derived rhFGF2 (e-FGF2) protein. Similar to the effects of e-FGF2, local delivery with p-FGF2 enhanced bone healing in the damaged region to higher levels than the ACS group. Exogenous addition of p-FGF2 or e-FGF2 exhibited similar effects on proliferation, mineralization, and osteogenic marker expression in MC3T3-E1 cells. Together, the current findings support the usefulness of this plant-based expression system for the production of biologically active rhFGF2.

SummaryPlants are attractive hosts for the production of recombinant glycoproteins for therapeutic use. Recent advances in glyco‐engineering facilitate the elimination of nonmammalian‐type glycosylation and introduction of missing pathways for customized N‐glycan formation. However, some therapeutically relevant recombinant glycoproteins exhibit unwanted truncated (paucimannosidic) N‐glycans that lack GlcNAc residues at the nonreducing terminal end. These paucimannosidic N‐glycans increase product heterogeneity and may affect the biological function of the recombinant drugs. Here, we identified two enzymes, β‐hexosaminidases (HEXOs) that account for the formation of paucimannosidic N‐glycans in Nicotiana benthamiana, a widely used expression host for recombinant proteins. Subcellular localization studies showed that HEXO1 is a vacuolar protein and HEXO3 is mainly located at the plasma membrane in N. benthamiana leaf epidermal cells. Both enzymes are functional and can complement the corresponding HEXO‐deficient Arabidopsis thaliana mutants. In planta expression of HEXO3 demonstrated that core α1,3‐fucose enhances the trimming of GlcNAc residues from the Fc domain of human IgG. Finally, using RNA interference, we show that suppression of HEXO3 expression can be applied to increase the amounts of complex N‐glycans on plant‐produced human α1‐antitrypsin.

Transgenic plant cell suspension culture systems have been utilized extensively as convenient and efficient expression systems for the production of recombinant human growth factors. We produced insulin-like growth factor-1 using a plant suspension culture system (p-IGF-1) and explored its effect on new bone formation in calvarial defects. We also compared the bone regenerating potential of p-IGF-1 with commercial IGF-1 derived from Escherichia coli (e-IGF-1). Male C57BL/6 mice underwent calvarial defect surgery, and the defects were loaded with absorbable collagen sponge (ACS) only (ACS group) or ACS impregnated with 13μg of p-IGF-1 (p-IGF-1 group) or e-IGF-1 (e-IGF-1 group). The sham group did not receive any treatment with ACS or IGFs after surgery. Live μCT and histological analyses showed critical-sized bone defects in the sham group, whereas greater bone formation was observed in the p-IGF-1 and e-IGF-1 groups than the ACS group both 5 and 10weeks after surgery. Bone mineral density, bone volume, and bone surface values were also higher in the IGF groups than in the ACS group. Local delivery of p-IGF-1 or e-IGF-1 more greatly enhanced the expression of osteoblast-specific markers, but inhibited osteoclast formation, in newly formed bone compared with ACS control group. Specifically, p-IGF-1 treatment induced higher expression of alkaline phosphatase, osteocalcin, and osteopontin in the defect site than did e-IGF-1. Furthermore, treatment with p-IGF-1, but not e-IGF-1, increased mineralization of MC3T3-E1 cells, with the attendant upregulation of osteogenic marker genes. Collectively, our findings suggest the potential of p-IGF-1 in promoting the processes required for bone regeneration.

The biochemical properties of Spirulina platensis in an internally illuminated photobioreactor (IlPBR) were investigated under different light-emitted diode (LED) wavelengths; blue (λ max = 450 and 460 nm), green (λ max = 525 nm), red (λ max = 630 and 660 nm), and white (6,500K), with various light intensities (200, 500, 1,000, and 2,000 μmol/m 2 /sec) were examined. The highest specific growth rate, maximum biomass, and phycocyanin productivity occurred under the red LEDs (0.39/day, 0.10 g/L/day, and 0.14 g/g-cell/day, respectively) at 1,000 μmol/m 2 /sec; the lowest growth rate was obtained under blue LEDs. Indeed, the size of trichomes was changed into short form under blue LEDs at all light intensities or all LEDs at 2,000 μmol/m 2 /sec for the first 2 days after inoculation, and S. platensis did not grow in the IlPBR under the dark condition. These results provide a base for different approaches for designing the pilot scale photobioreactor and developing cost-effective light sources.

ABSTRACTSoftware is commonly built from reusable components that provide desired functionalities. Although component reuse significantly improves software productivity, insecure component usage can lead to security vulnerabilities in client applications. For example, we noticed that widely-used IE-based browsers, such as IE Tab, do not enable important security features that IE enables by default, even though they all use the same browser components. This insecure usage renders these IE-based browsers vulnerable to the attacks blocked by IE. To our knowledge, this important security aspect of component reuse has largely been unexplored. This paper presents the first practical framework for detecting and analyzing vulnerabilities of insecure component usage. Its goal is to enforce and support secure component reuse. Our core approach is based on differential testing and works as follows. Suppose that component C maintains a security policy configuration to block certain malicious behavior. If two clients of component C, say a reference and a test subject, handle the malicious behavior inconsistently, the test subject uses C insecurely. In particular, we model component usage related to a policy based on 1) accesses to the configuration state inside the component and 2) the conditional jumps affected by the data read from the state. We utilize this model to detect inconsistent policy evaluations, which can lead to insecure component usage. We have implemented our technique for Windows applications and used it to detect and analyze insecure usage of popular software components. Our evaluation results show that 1) insecure component usage is a general concern and frequently occurs in widely-used software, and 2) our detection framework is practical and effective at detecting and analyzing insecure component usage. In particular, it detected several serious, new vulnerabilities and helped perform detailed analysis of insecure component usage. We have reported these to the affected software vendors, some of whom have already acknowledged our findings and are actively addressing them.

Dynamic loading of software components (e.g., libraries or modules) is a widely used mechanism for an improved system modularity and flexibility. Correct component resolution is critical for reliable and secure software execution. However, programming mistakes may lead to unintended or even malicious components being resolved and loaded. In particular, dynamic loading can be hijacked by placing an arbitrary file with the specified name in a directory searched before resolving the target component. Although this issue has been known for quite some time, it was not considered serious because exploiting it requires access to the local file system on the vulnerable host. Recently, such vulnerabilities have started to receive considerable attention as their remote exploitation became realistic. It is now important to detect and fix these vulnerabilities. In this paper, we present the first automated technique to detect vulnerable and unsafe dynamic component loadings. Our analysis has two phases: 1) apply dynamic binary instrumentation to collect runtime information on component loading (online phase), and 2) analyze the collected information to detect vulnerable component loadings (offline phase). For evaluation, we implemented our technique to detect vulnerable and unsafe component loadings in popular software on Microsoft Windows and Linux. Our evaluation results show that unsafe component loading is prevalent in software on both OS platforms, and it is more severe on Microsoft Windows. In particular, our tool detected more than 4,000 unsafe component loadings in our evaluation, and some can lead to remote code execution on Microsoft Windows.

Software is commonly built from reusable components that provide desired functionalities. Although component reuse significantly improves software productivity, insecure component usage can lead to security vulnerabilities in client applications. For example, we noticed that widely-used IE-based browsers, such as IE Tab, do not enable important security features that IE enables by default, even though they all use the same browser components. This insecure usage renders these IE-based browsers vulnerable to the attacks blocked by IE. To our knowledge, this important security aspect of component reuse has largely been unexplored. This paper presents the first practical framework for detecting and analyzing vulnerabilities of insecure component usage. Its goal is to enforce and support secure component reuse. Our core approach is based on differential testing and works as follows. Suppose that component C maintains a security policy configuration to block certain malicious behavior. If two clients of component C, say a reference and a test subject, handle the malicious behavior inconsistently, the test subject uses C insecurely. In particular, we model component usage related to a policy based on 1) accesses to the configuration state inside the component and 2) the conditional jumps affected by the data read from the state. We utilize this model to detect inconsistent policy evaluations, which can lead to insecure component usage. We have implemented our technique for Windows applications and used it to detect and analyze insecure usage of popular software components. Our evaluation results show that 1) insecure component usage is a general concern and frequently occurs in widelyused software, and 2) our detection framework is practical and effective at detecting and analyzing insecure component usage. In particular, it detected several serious, new vulnerabilities and helped perform detailed analysis of insecure component usage. We have reported these to the affected software vendors, some of whom have already acknowledged our findings.

Dynamic loading of software components is a commonly used mechanism to achieve better flexibility and modularity in software. For an application’s runtime safety, it is important for the application to load only its intended components. However, programming mistakes may lead to failures to load a component, or even worse, to load a malicious component. Recent work has shown that these errors are both prevalent and severe, sometimes leading to remote code execution attacks. The work is based on dynamic analysis by monitoring and analyzing runtime component loadings. Although simple and effective in detecting real errors, it suffers from limited code coverage and may miss important vulnerabilities. Thus, it is desirable to develop effective techniques to detect all possible unsafe component loadings. This paper presents the first static binary analysis aiming at detecting all possible loading-related errors. The key challenge is how to scalably and precisely compute what components may be loaded at relevant program locations. Our main insight is that this information is often determined locally from the component loading call sites. This motivates us to design a demand-driven analysis, working backward starting from the relevant call sites. In particular, for a given call site c, we first compute its context-sensitive executable slices, one for each execution context. Then we emulate the slices to obtain the set of components possibly loaded at c. This novel combination of slicing and emulation achieves good scalability and precision by avoiding expensive symbolic analysis. We implemented our technique and evaluated its effectiveness against the existing dynamic technique on nine popular Windows applications. Results show that our tool has better coverage and is precise—it is able to detect many more unsafe loadings. It is also scalable and finishes analyzing all nine applications within minutes.

The analysis of software similarity has many applications such as detecting code clones, software plagiarism, code theft, and polymorphic malware. Because often source code is unavailable and code obfuscation is used to avoid detection, there has been much research on developing effective models to capture runtime behavior to aid detection. Existing models focus on low-level information such as dependency or purely occurrence of function calls, and suffer from poor precision, poor scalability, or both. To overcome limitations of existing models, this paper introduces a precise and succinct behavior representation that characterizes high-level object-accessing patterns as regular expressions. We first distill a set of high-level patterns (the alphabet S of the regular language) based on two pieces of information: function call patterns to access objects and type state information of the objects. Then we abstract a runtime trace of a program P into a regular expression e over the pattern alphabet S to produce P's behavior signature. We show that software instances derived from the same code exhibit similar behavior signatures and develop effective algorithms to cluster and match behavior signatures. To evaluate the effectiveness of our behavior model, we have applied it to the similarity analysis of polymorphic malware. Our results on a large malware collection demonstrate that our model is both precise and succinct for effective and scalable matching and detection of polymorphic malware.

A synthetic bovine trypsinogen (sbTrypsinogen) was synthesized on the basis of rice-optimized codon usage via an overlap PCR strategy, prior to being expressed under the control of the sucrose starvation-inducible rice α-amylase 3D (RAmy3D) promoter. Secretion of trypsin into the culture medium was achieved by using the existing signal peptide. The plant expression vector was introduced into rice calli (Oryza sativa L. cv. Dongjin), mediated by Agrobacterium tumefaciens. The integration of the sbTrypsinogen gene into the chromosome of the transgenic rice callus was verified via genomic DNA PCR amplification, and sbTrypsin expression in transgenic rice suspension cells was confirmed via Northern blot analysis. Western blot analysis detected glycosylated proteins in the culture medium, having masses from 24 to 26 kDa, following induction by sugar starvation. Proteolytic activity of the rice-derived trypsin was confirmed by gelatin zymogram, and was similar to that of the commercial bovine-produced trypsin. The yields of sbTrypsin that accumulated in the transgenic rice cell suspension medium were 15 mg/L at 5 days after sugar starvation.