Delivery of protein antigens to the immune system by fusion-active virosomes: a comparison with liposomes and ISCOMs

The induction of effective cellular and humoral immune responses against protein antigens is of major importance in vaccination strategies against infectious diseases and cancer. Immunization with protein alone in general does not result in efficient induction of cytotoxic T lymphocyte (CTL) and antibody responses. Numerous other immunization strategies have been explored. In this review we will discuss a number of lipid-based antigen delivery systems suitable for the induction of CTL responses. These systems comprise reconstituted virus envelopes (virosomes), liposomes, and immune-stimulating complexes (ISCOMs). We will concentrate on delivery of the protein antigen ovalbumin (OVA) since extensive studies with this antigen have been performed for all of the systems discussed, allowing direct comparison of antigen delivery efficiency. Stimulation of CTL activity requires processing of the antigen in the cytosol of antigen-presenting cells (APCs) and presentation of antigenic peptides on surface major histocompatibility class I complexes (MHC class I). In vitro, the ability of antigen delivery systems to induce MHC class I presentation indeed correlates with their capacity to deliver antigen to the cytosol of cells. This capacity appears to be less important for the induction of cytotoxic T lymphocytes in vivo. Instead, other properties of the antigen delivery system like activation of APCs and induction of T helper cells play a more prominent role. Fusion-active virosomes appear to be a very potent system for induction of CTL activity, most likely since virosomes combine efficient delivery of antigen with general stimulation of the immune system.     

Prime/boost immunotherapy of HPV16-induced tumors with E7 protein delivered by Bordetella adenylate cyclase and modified vaccinia virus Ankara

The Bordetella adenylate cyclase toxoid (CyaA) targets cells expressing the α_Mβ₂ integrin receptor CD11b/CD18 (CR3 or Mac-1) and can penetrate into cytosol of professional antigen-presenting cells, such as dendritic cells. This allows us to use CyaA for delivery of passenger antigens into the cytosolic pathway of processing and MHC class I-restricted presentation, which can promote induction of antigen-specific CD8⁺ cytotoxic T-lymphocyte immune responses. We show here that vaccination with a genetically detoxified CyaA336/E7 protein, carrying the full-length oncoprotein E7 of the human papilloma virus 16 inserted at position 336 of the cell-invasive AC domain of CyaA, induces an E7-specific CD8⁺ T-cell immune response and confers on mice protective, as well as therapeutic immunity against challenge with TC-1 tumor cells expressing the E7 oncoprotein. The therapeutic efficacy of priming with the CyaA336/E7 vaccine could further be enhanced by a heterologous booster immunization with a highly attenuated modified vaccinia virus Ankara (MVA) expressing the E7 protein fused to the lysosome-associated membrane protein (LAMP1). These results establish the potential of CyaA as a new antigen delivery tool for prime/boost immunotherapy of tumors. This paper won the poster prize at the conference “Progress in Vaccination against Cancer 4”, PIVAC 4, held in Freudenstadt-Lauterbad, Black Forest, Germany, from 22 to 25 September 2004. For further material on this conference, please see the series of Symposium Papers, published     

What is needed for effective antitumor immunotherapy? Lessons learned using Listeria monocytogenes as a live vector for HPV-associated tumors

As a vaccine vector, Listeria monocytogenes targets the innate immune system, resulting in a cytokine response that enhances antigen-presenting cell function as well as inducing a Th1 profile. It also enhances cell-mediated immunity by targeting antigen delivery in antigen-presenting cells to both the MHC class I pathway of exogenous presentation that activates CD8 T cells and the MHC class II pathway that processes antigen endogenously and presents it to CD4 T cells. In this review, we describe the development of vaccine constructs that target the human papillomavirus 16 (HPV-16) E7 antigen, and we characterize their effects on tumor regression as well as various immune parameters both innate and adaptive. In particular, we describe the effect on tumor angiogenesis, induction of antitumor suppressor factors like CD4⁺CD25⁺ T cells and regulatory cytokines TGF- and IL-10, homing and infiltration of antigen-specific CD8⁺ T cells to the tumor, and also effects of the vaccines on antigen-presenting cells, especially focusing on dendritic cell maturation and ability to influence tumor regression. We believe that the identification of several immune parameters that correlate with antitumor efficacy, and of some that have a negative correlation, may have wider application for other cancer immunotherapeutic approaches.This article is a symposium paper from the second international conference Strategies for Immune Therapy, 29 February–3 March 2004, Würzburg, Germany; summarized by G. Pawelec and C. Gouttefangeas.     

Cell-penetrating peptides: Application in vaccine delivery

Recent years have seen a surge in interest in cell-penetrating peptides (CPP) as an efficient means for delivering therapeutic targets into cellular compartments. The cell membrane is impermeable to hydrophilic substances yet linking to CPP can facilitate delivery into cells. Thus the unique translocatory property of CPP ensures they remain an attractive carrier, with the capacity to deliver cargoes in an efficient manner having applications in drug delivery, gene transfer and DNA vaccination. Fundamental for an effective vaccine is the delivery of antigen epitopes to antigen-presenting cells, ensuing processing and presentation and induction of an immune response. Vaccination with proteins or synthetic peptides incorporating CTL epitopes have proven limited due to the failure for exogenous antigens to be presented efficiently to T cells. Linking of antigens to CPP overcomes such obstacles by facilitating cellular uptake, processing and presentation of exogenous antigen for the induction of potent immune responses. This review will encompass the various strategies for the delivery of whole proteins, T cell epitopes and preclinical studies utilizing CPP for cancer vaccines.     

Antibodies engineered with IgD specificity efficiently deliver integrated T-cell epitopes for antigen presentation by B cells.

We have developed a strategy for improving the stimulation of T cells during immune responses by constructing recombinant antibodies that enhance the delivery of antigen to antigen-presenting cells, such as B cells. These antibodies have variable regions specific for surface molecules on B cells, and a constant region with an inserted antigen. In vitro, such antibodies make B cells approximately 1000-fold more efficient at presenting antigen and stimulating specific T cells. In vivo, the antibodies turn B cells of the spleen into potent stimulators of T cells. This approach may be useful for the generation of new vaccines.     

Binding of antigen in the absence of histocompatibility proteins by arsonate-reactive T-cell clones

Inducer T-cell clones reactive to the p-azobenzenearsonate (arsonate) hapten possess binding sites for radioactive arsanylated proteins, which are not present on clones with other antigen specificities. Binding occurred in the absence of histocompatibility proteins. Binding was specific for the p-azobenzenearsonate hapten, since unconjugated proteins and proteins conjugated to the nonactivating o-azobenzenearsonate hapten neither bound to the clones nor competed binding of radioactive antigen. One of the clones was studied in more detail, using a panel of structural analogs of arsonate conjugated to the carrier protein ovalbumin. All conjugates that activated the clone in the presence of antigen-presenting cells also competed binding of radioactive antigen in the absence of antigen-presenting cells. Nonactivating conjugates did not compete binding. Based on evidence in this and the succeeding paper (Rao et al., accompanying paper), we suggest that these arsonate-binding sites may include the physiological antigen receptors of arsonate-reactive T-cell clones.     

Plant-derived protein bodies as delivery vehicles for recombinant proteins into mammalian cells

The encapsulation of biopharmaceuticals into micro- or nanoparticles is a strategy frequently used to prevent degradation or to achieve the slow release of therapeutics and vaccines. Protein bodies (PBs), which occur naturally as storage organelles in seeds, can be used as such carrier vehicles. The fusion of the N-terminal sequence of the maize storage protein, γ-zein, to other proteins is sufficient to induce the formation of PBs, which can be used to bioencapsulate recombinant proteins directly in the plant production host. In addition, the immunostimulatory effects of zein have been reported, which are advantageous for vaccine delivery. However, little is known about the interaction between zein PBs and mammalian cells. To better understand this interaction, fluorescent PBs, resulting from the fusion of the N-terminal portion of zein to a green fluorescent protein, was produced in Nicotiana benthamiana leaves, recovered by a filtration-based downstream procedure, and used to investigate their internalization efficiency into mammalian cells. We show that fluorescent PBs were efficiently internalized into intestinal epithelial cells and antigen-presenting cells (APCs) at a higher rate than polystyrene beads of comparable size. Furthermore, we observed that PBs stimulated cytokine secretion by epithelial cells, a characteristic that may confer vaccine adjuvant activities through the recruitment of APCs. Taken together, these results support the use of zein fusion proteins in developing novel approaches for drug delivery based on controlled protein packaging into plant PBs.     

Bacterial systems for the delivery of eukaryotic antigen expression vectors

Attenuated bacterial strains allow the administration of recombinant vaccines via the mucosal surfaces. Whereas attenuated bacteria are generally engineered to express heterologous antigens, a novel approach employs intracellular bacteria for the delivery of eukaryotic antigen expression vectors (so-called DNA vaccines). This strategy allows a direct delivery of DNA to professional antigen-presenting cells (APC), such as macrophages and dendritic cells (DC), through bacterial infection. The bacteria used for DNA vaccine delivery either enter the host cell cytosol after phagocytosis by the APC, for example, Shigella and Listeria, or they remain in the phagosomal compartment, such as Salmonella. Both intracellular localizations of the bacterial carriers seem to be suitable for successful delivery of DNA vaccine vectors.     

Whole-animal Imaging and Flow Cytometric Techniques for Analysis of Antigen-specific CD8+ T Cell Responses after Nanoparticle Vaccination

Traditional vaccine adjuvants, such as alum, elicit suboptimal CD8+ T cell responses. To address this major challenge in vaccine development, various nanoparticle systems have been engineered to mimic features of pathogens to improve antigen delivery to draining lymph nodes and increase antigen uptake by antigen-presenting cells, leading to new vaccine formulations optimized for induction of antigen-specific CD8+ T cell responses. In this article, we describe the synthesis of a “pathogen-mimicking” nanoparticle system, termed interbilayer-crosslinked multilamellar vesicles (ICMVs) that can serve as an effective vaccine carrier for co-delivery of subunit antigens and immunostimulatory agents and elicitation of potent cytotoxic CD8+ T lymphocyte (CTL) responses. We describe methods for characterizing hydrodynamic size and surface charge of vaccine nanoparticles with dynamic light scattering and zeta potential analyzer and present a confocal microscopy-based procedure to analyze nanoparticle-mediated antigen delivery to draining lymph nodes. Furthermore, we show a new bioluminescence whole-animal imaging technique utilizing adoptive transfer of luciferase-expressing, antigen-specific CD8+ T cells into recipient mice, followed by nanoparticle vaccination, which permits non-invasive interrogation of expansion and trafficking patterns of CTLs in real time. We also describe tetramer staining and flow cytometric analysis of peripheral blood mononuclear cells for longitudinal quantification of endogenous T cell responses in mice vaccinated with nanoparticles.     

Apoptosis facilitates antigen presentation to T lymphocytes through MHC-I and CD1 in tuberculosis

Protective immunity against Mycobacterium tuberculosis involves major histocompatibility complex class I (MHC-I)- and CD1-restricted CD8 T cells, but the mechanisms underlying antigen delivery to antigen-presenting molecules remain enigmatic. Macrophages, the primary host cells for mycobacteria, are CD1-negative. Here we show that M. tuberculosis phagosomes are secluded from the cytosolic MHC-I processing pathway and that mycobacteria-infected cells lose their antigen-presenting capacity. We also show that mycobacteria induce apoptosis in macrophages, causing the release of apoptotic vesicles that carry mycobacterial antigens to uninfected antigen-presenting cells (APCs). Inhibition of apoptosis reduced transfer of antigens to bystander cells and activation of CD8 T cells. Uninfected dendritic cells, which engulfed extracellular vesicles, were indispensable for subsequent cross-presentation of antigens, through MHC-I and CD1b, to T cells from mycobacteria-sensitized donors. This new 'detour' pathway for presentation of antigens from a phagosome-contained pathogen shows the functional significance of infection-induced apoptosis in the activation of CD8 T cells specific for both protein and glycolipid antigens in tuberculosis.     

Gene therapy for immunologic tolerance: using bone marrow-derived cells to treat autoimmunity and hemophilia

Bone marrow derived cells, especially B lymphocytes, have been shown to function as tolerogenic antigen-presenting cells (APC's) both in vivo and in vitro. In addition, it is well established that immunoglobulins can function as potent tolerogenic carriers for associated epitopes. We have taken advantage of these properties to develop a gene therapy approach to induce unresponsiveness in a number of animal models for clinical diseases. In our system, we engineered target peptide-IgG constructs into retroviral vectors and transduced hematopoietic cells to create tolerogenic antigen-presenting cells. In this review, we discuss the strategies and mechanism of our gene therapy approach mediated by B cells, as well as by bone marrow cells, for tolerance acquisition in various mouse models for autoimmune disease and hemophilia A. Our results show that MHC class II and co-stimulatory molecules must be expressed on the tolerogenic antigen presenting cells for efficacy. This therapy requires regulatory T cells for both the induction and maintenance of tolerance. The putative role of epitopes provided by the IgG carrier in this process. Studies in non-human primates and with human T cell clones in vitro are in progress to transition this approach to the clinic. The use of stem cells and B cell-delivered gene therapy in human clinical diseases may soon become a reality.     

Transcutaneous antigen delivery system

Transcutaneous immunization refers to the topical application of antigens onto the epidermis. Transcutaneous immunization targeting the Langerhans cells of the skin has received much attention due to its safe, needle-free, and noninvasive antigen delivery. The skin has important immunological functions with unique roles for antigen-presenting cells such as epidermal Langerhans cells and dermal dendritic cells. In recent years, novel vaccine delivery strategies have continually been developed; however, transcutaneous immunization has not yet been fully exploited due to the penetration barrier represented by the stratum corneum, which inhibits the transport of antigens and adjuvants. Herein we review recent achievements in transcutaneous immunization, focusing on the various strategies for the enhancement of antigen delivery and vaccination efficacy. [BMB Reports 2013; 46(1): 17-24]     

Liposome-Mediated Cytoplasmic Delivery of Proteins: An Effective Means of Accessing the MHC Class I-Restricted Antigen Presentation Pathway

Different types of liposomes have been employed to deliver soluble antigen for processing and presentation in the major histocompatibility complex class I-restricted pathway. Anionic pH-sensitive liposomes as well as cationic liposomes efficiently sensitize antigen-presenting cells for recognition by the class I-restricted cytotoxic T lymphocytes (CTL). Cytoplasmic delivery of liposome-entrapped antigen from an endocytic compartment allows the exogenous antigen to gain access to the class I presentation pathway. Cytoplasmic delivery, however, is probably not the only mechanism by which liposomes induce the class I-restricted CTL priming in vivo. Macrophages play a central role in the processing of the liposome-encapsulated antigens. The processed antigen fragments are probably released by the macrophages and taken up by the nearby dendritic cells for antigen presentation. Collaboration between the two types of immune cells with the help of the appropriate costimulatory factors is the central theme for this hypothesis. In this case, the host immune system utilizes the similar mechanism for other membranous, particulate antigens to process and present the liposomal antigens.     

Synthesis and assembly of dengue virus envelope protein fused to cholera toxin B subunit into biologically active oligomers in transgenic tomato (<Emphasis Type="Italic">Solanum lycopersicum</Emphasis>)

Dengue is the fastest growing mosquito-borne disease worldwide, causing nearly 400 million infections annually. A universally applicable dengue virus vaccine is required to arrest its spread. Here, we generated an edible dengue vaccine by expressing the dengue fusion protein in tomatoes, which is a desirable expression system owing to the inherent adjuvanticity of alpha tomatine and immunogenicity of the tomato lectin/microbial antigen complex. The B subunit of Vibrio cholera toxin (CTB) was genetically fused to dengue envelope antigen for improved delivery to antigen-presenting cells and enhanced immunogenicity, while avoiding immunological tolerance. We utilized domain III of the dengue envelope protein (EDIII), as it has been shown to induce serotype-specific neutralizing antibodies. The CTB–EDIII fusion gene construct containing an endoplasmic reticulum target sequence was introduced into tomato plants by Agrobacterium tumefaciens-mediated gene transformation, and the expression of CTB–EDIII in transgenic plants was confirmed by DNA, RNA and protein analyses. Accumulated fusion protein accounted for up to 0.015 % of total soluble protein, and it assembled into fully functional pentamers as demonstrated by binding to GM1 ganglioside. Future work will involve testing of transgenic tomatoes for immunogenicity in mice following oral delivery.     

需钠弧菌外膜囊泡的蛋白质组分析与异源蛋白的递送

[背景] 需钠弧菌（Vibrio natriegens）是一种快速生长的革兰氏阴性菌,作为一种新兴工具在生物技术领域有重要的应用潜力。此前的研究主要集中在开发利用V. natriegens成为体内外重组蛋白生产的工具。然而,许多支持细菌进行快速生长和蛋白质生产的生理活动大部分仍未确定。外膜囊泡（Outer Membrane Vesicle,OMV）是由革兰氏阴性细菌普遍产生的一种球形小泡,其不仅具有重要的功能,而且还可以作为一种应用于疫苗治疗的高效运载工具。[目的] 表征指数生长期OMV的蛋白质组并利用OMV进行异源蛋白的递送。[方法] 使用透射电镜、动态光散射和质谱学的方法,观察OMV的形态及粒径分布并鉴定蛋白组成。以超折叠绿色荧光蛋白（Superfolded Green Fluorescent Protein,sfGFP）作为货物蛋白来确定OMV蛋白载体。[结果] 从细菌培养的指数期中期和末期分别提取的OMV中鉴定到了288个和317个蛋白。这些蛋白分属不同的功能组,包括ABC转运蛋白、鞭毛、双组分系统。相比之下,同时鉴定了全细胞样品,其在指数期中期和末期分别含有1 480个和1 565个蛋白。我们筛选OMV的蛋白作为候选载体发现了一种属于OmpA家族的蛋白（命名为OmpA24）,其能够将sfGFP以融合货物蛋白的形式运载到OMV中。[结论] 首次证实V. natriegens能够在指数生长期产生OMV,并展示了第一个不同生长时期OMV和全细胞的蛋白质组鉴定结果。OmpA24是将外源融合货物蛋白呈递到OMV中的有前景的载体。本研究有助于促进V. natriegens在蛋白表达和OMV介导的分泌中的应用。     

Bacterial delivery of functional messenger RNA to mammalian cells

The limited access to the nuclear compartment may constitute one of the major barriers after bacteria-mediated expression plasmid DNA delivery to eukaryotic cells. Alternatively, a self-destructing Listeria monocytogenes strain was used to release translation-competent mRNA directly into the cytosol of epithelial cells, macrophages and human dendritic cells. Enhanced green fluorescent protein (EGFP)-encoding mRNA, adapted for translation in mammalian cells by linking an IRES element to the 5'-end of the egfp coding sequence, was produced by T7 RNA polymerase in the carrier bacteria upon entry into the cytosol where the mRNA is efficiently released from the lysed bacteria and immediately translated in eukaryotic host cells. Besides the much earlier expression of EGFP being detectable already 4 h after infection, the number of EGFP expressing mammalian cells obtained with this novel RNA delivery technique is comparable to or - especially in phagocytic cells - even higher than that obtained with the expression plasmid DNA delivery strategy. Accordingly, bacteria-mediated delivery of ovalbumin-encoding mRNA to macrophages resulted in efficient antigen processing and presentation in vitro indicating that this approach may also be adapted for the in vivo delivery of antigen-encoding mRNA leading to a more efficient immune response when applied to vaccine development.     

Inhibition of macropinocytosis blocks antigen presentation of type II collagen in vitro and in vivo in HLA-DR1 transgenic mice

Professional antigen-presenting cells, such as dendritic cells, macrophages and B cells have been implicated in the pathogenesis of rheumatoid arthritis, constituting a possible target for antigen-specific immunotherapy. We addressed the possibility of blocking antigen presentation of the type II collagen (CII)-derived immunodominant arthritogenic epitope CII_259–273 to specific CD4 T cells by inhibition of antigen uptake in HLA-DR1-transgenic mice in vitro and in vivo. Electron microscopy, confocal microscopy, subcellular fractionation and antigen presentation assays were used to establish the mechanisms of uptake, intracellular localization and antigen presentation of CII by dendritic cells and macrophages. We show that CII accumulated in membrane fractions of intermediate density corresponding to late endosomes. Treatment of dendritic cells and macrophages with cytochalasin D or amiloride prevented the intracellular appearance of CII and blocked antigen presentation of CII_259–273 to HLA-DR1-restricted T cell hybridomas. The data suggest that CII was taken up by dendritic cells and macrophages predominantly via macropinocytosis. Administration of amiloride in vivo prevented activation of CII-specific polyclonal T cells in the draining popliteal lymph nodes. This study suggests that selective targeting of CII internalization in professional antigen-presenting cells prevents activation of autoimmune T cells, constituting a novel therapeutic strategy for the immunotherapy of rheumatoid arthritis.     

Polymer based cardiovascular gene therapy

Therapeutic angiogenesis is a new potential treatment in cardiovascular disease. It is performed by the delivery of the angiogenic agents (protein, gene). Most important consideration for gene therapy is the construction of an effective therapeutic gene. Currently, VEGF is the most effective therapeutic gene for the neovascularization. We constructed the hypoxia-regulated VEGF plasmid using the Epo enhancer and RTP801 promoter. The efficiency of the pEpo-SV-VEGF and pRTP801-VEGF were evaluated by various methodsin vitro andin vivo. The results suggested that the hypoxia-inducible VEGF gene therapy system is effective and safe, which may be useful for the gene therapy of ischemic heart disease. Development of a safe and efficient gene carrier is another main requirement for successful gene therapy. Although viralbased gene delivery is currently the most effective way to transfer genes to cells, nonviral vectors are increasingly being considered forin vivo gene delivery. The advantages of nonviral gene therapy are lack of specific immunogenecity, simplicity of use, and ease of large-scale production. In addition, the simple conjugation of a targeting moiety to nonviral gene carrier can facilitate tissue-targeting gene delivery. We have developed two new gene carrier systems, TerplexDNA and WSLP (water soluble lipopolymer). These two are efficient carrier to ischemic myocardium and has low toxicity and high transfection efficiency. So it may allow for application ofin vivo gene therapy in the treatment of heart disease.     

Plant heat shock protein 70 as carrier for immunization against a plant-expressed reporter antigen

Mammalian Heat Shock Proteins (HSP), have potent immune-stimulatory properties due to the natural capability to associate with polypeptides and bind receptors on antigen presenting cells. The present study was aimed to explore whether plant HSP, and in particular HSP70, share similar properties. We wanted in particular to evaluate if HSP70 extracted in association to naturally bound polypeptides from plant tissues expressing a recombinant “reporter” antigen, carry antigen-derived polypeptides and can be used to activate antigen-specific immune responses. This application of HSP70 has been very poorly investigated so far. The analysis started by structurally modeling the plant protein and defining the conditions that ensure maximal expression levels and optimal recovery from plant tissues. Afterwards, HSP70 was purified from Nicotiana benthamiana leaves transiently expressing a heterologous “reporter” protein. The purification was carried out taking care to avoid the release from HSP70 of the polypeptides chaperoned within plant cells. The evaluation of antibody titers in mice sera subsequent to the subcutaneous delivery of the purified HSP70 demonstrated that it is highly effective in priming humoral immune responses specific to the plant expressed “reporter” protein. Overall results indicated that plant-derived HSP70 shares structural and functional properties with the mammalian homologue. This study paves the way to further investigations targeted at determining the properties of HSP70 extracted from plants expressing foreign recombinant antigens as a readily available immunological carrier for the efficient delivery of polypeptides derived from these antigens.     

Expression of cholera toxin B subunit–lumbrokinase in edible sunflower seeds–the use of transmucosal carrier to enhance its fusion protein's effect on protection of rats and mice against thrombosis

Lumbrokinase (LK) is a group of serine proteases with strong fibrinolytic and thrombolytic activities and is useful for treating diseases caused by thrombus. Cholera toxin B subunit (CTB) has been widely used to facilitate antigen delivery by serving as an effective mucosal carrier molecule for the induction of oral tolerance. We investigate here the application of CTB as a transmucosal carrier in enhancing its fusion protein‐LKs effect to protect rats against thrombosis. Thus, in this study, CTB‐LK fusion gene separated by a furin cleavage site was expressed in seeds of Helianthus annuus L. The activity of recombinant protein in seeds of transgenic sunflower was confirmed by Western blot analysis, fibrin plate assays and G_M1‐ganglioside ELISA. The thrombosis model of rats and mice revealed that the oral administration of peeled seeds of sunflower expressing CTB‐LK had a more significant anti‐thrombotic effect on animals compared with that administration of peeled seeds of sunflower expressing LK. It is possible to conclude that CTB can successfully enhance its fusion protein to be absorbed in rats or mice thrombosis model. The use of CTB as a transmucosal carrier in the delivery of transgenic plant‐derived oral therapeutic proteins was supported. In addition, for the purpose of that recombinant CTB‐LK was designed for oral administration, thus the expression of CTB‐LK in edible sunflower seeds eliminated the need for downstream processing of proteins. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1029–1039, 2014