Recombinant MBP-pσ1 expressed in soybean seeds delays onset and reduces developing disease in an animal model of multiple sclerosis.

It is estimated that multiple sclerosis (MS) affects over 2.8 million people worldwide, with a prevalence that is expected to continue growing over time. Unfortunately, there is no cure for this autoimmune disease. For several decades, antigen-specific treatments have been used in animal models of experimental autoimmune encephalomyelitis (EAE) to demonstrate their potential for suppressing autoimmune responses. Successes with preventing and limiting ongoing MS disease have been documented using a wide variety of myelin proteins, peptides, autoantigen-conjugates, and mimics when administered by various routes. While those successes were not translatable in the clinic, we have learned a great deal about the roadblocks and hurdles that must be addressed if such therapies are to be useful. Reovirus sigma1 protein (pσ1) is an attachment protein that allows the virus to target M cells with high affinity. Previous studies showed that autoantigens tethered to pσ1 delivered potent tolerogenic signals and diminished autoimmunity following therapeutic intervention. In this proof-of-concept study, we expressed a model multi-epitope autoantigen (human myelin basic protein, MBP) fused to pσ1 in soybean seeds. The expression of chimeric MBP-pσ1 was stable over multiple generations and formed the necessary multimeric structures required for binding to target cells. When administered to SJL mice prophylactically as an oral therapeutic, soymilk formulations containing MBP-pσ1 delayed the onset of clinical EAE and significantly reduced developing disease. These results demonstrate the practicality of soybean as a host for producing and formulating immune-modulating therapies to treat autoimmune diseases.

Systemic cytokine and chemokine responses in immunized mice challenged with staphylococcal enterotoxin B.

The cytokine storm induced by staphylococcal enterotoxin B (SEB) describes the rapid and dramatic induction of mediators which are likely responsible for the toxin's deleterious effects. However despite the use of numerous animal models for investigating SEB related illness in humans, mechanisms of toxicity and correlates of protection remain unclear. In the present study, we used an LPS-potentiated model of SEB lethality to investigate the toxin-induced cytokine and chemokine responses in untreated and immunized mice. Of 30 separate mediators analyzed, serum levels for 28 or 27 of these cytokines and chemokines were elevated following administration of dosages of 3 or 30 LD50 of native SEB, respectively. Mice immunized with a non-toxic SEB vaccine candidate expressed in either E. coli or transgenic soy expression systems were protected from lethality when challenged with potentiated SEB. The majority of SEB-induced cytokines and chemokines (21 of 28 or 23 of 27 following challenge with dosages of 3 or 30 LD50 of native SEB, respectively) were significantly decreased in mice immunized with an SEB vaccine candidate when compared to control animals. Together, these studies provide the most comprehensive evaluation of the cytokine storm induced in this LPS-potentiated model of SEB lethality to date. As with other animal models, the identification of those mediators which are necessary and sufficient for SEB-induced toxicity remains unclear.

Transcript Polymorphism Rates in Soybean Seed Tissue Are Increased in a Single Transformant of Glycine max.

Transgenic crops have been utilized for decades to enhance agriculture and more recently have been applied as bioreactors for manufacturing pharmaceuticals. Recently, we investigated the gene expression profiles of several in-house transgenic soybean events, finding one transformant group to be consistently different from our controls. In the present study, we examined polymorphisms and sequence variations in the exomes of the same transgenic soybean events. We found that the previously dissimilar soybean line also exhibited markedly increased levels of polymorphisms within mRNA transcripts from seed tissue, many of which are classified as gene expression modifiers. The results from this work will direct future investigations to examine novel SNPs controlling traits of great interest for breeding and improving transgenic soybean crops. Further, this study marks the first work to investigate SNP rates in transgenic soybean seed tissues and demonstrates that while transgenesis may induce abundant unanticipated changes in gene expression and nucleotide variation, phenotypes and overall health of the plants examined remained unaltered.

A Sublethal Swine Model for Defining In Vivo Superantigen-Induced Responses Following Exposure to Staphylococcal Enterotoxin B.

In vivo responses to bacterially derived superantigen-like toxins have been difficult to define due to the inherent limitations with rodent models and the relevance that the results obtained from such models may, or may not, have for human pathophysiology. Further the use of challenge doses of superantigen toxins that are lethal or supra-lethal complicates analogies to human exposures which are rarely fatal. Here, we utilize the superantigen, staphylococcal enterotoxin B, at doses that are sublethal in a swine model of toxin-induced incapacitation. Relevant dosing using an animal species for which this toxin is a true superantigen distinguishes this model.

CONTRAILS: A tool for rapid identification of transgene integration sites in complex, repetitive genomes using low-coverage paired-end sequencing.

Transgenic crops have become a staple in modern agriculture, and are typically characterized using a variety of molecular techniques involving proteomics and metabolomics. Characterization of the transgene insertion site is of great interest, as disruptions, deletions, and genomic location can affect product selection and fitness, and identification of these regions and their integrity is required for regulatory agencies. Here, we present CONTRAILS (Characterization of Transgene Insertion Locations with Sequencing), a straightforward, rapid and reproducible method for the identification of transgene insertion sites in highly complex and repetitive genomes using low coverage paired-end Illumina sequencing and traditional PCR. This pipeline requires little to no troubleshooting and is not restricted to any genome type, allowing use for many molecular applications. Using whole genome sequencing of in-house transgenic Glycine max, a legume with a highly repetitive and complex genome, we used CONTRAILS to successfully identify the location of a single T-DNA insertion to single base resolution.

A Comparison of transgenic and wild type soybean seeds: analysis of transcriptome profiles using RNA-Seq.

BACKGROUND: Soybean (Glycine max) has been bred for thousands of years to produce seeds rich in protein for human and animal consumption, making them an appealing bioreactor for producing valuable recombinant proteins at high levels. However, the effects of expressing recombinant protein at high levels on bean physiology are not well understood. To address this, we investigated whether gene expression within transgenic soybean seed tissue is altered when large amounts of recombinant proteins are being produced and stored exclusively in the seeds. We used RNA-Seq to survey gene expression in three transgenic soybean lines expressing recombinant protein at levels representing up to 1.61 % of total protein in seed tissues. The three lines included: ST77, expressing human thyroglobulin protein (hTG), ST111, expressing human myelin basic protein (hMBP), and 764, expressing a mutant, nontoxic form of a staphylococcal subunit vaccine protein (mSEB). All lines selected for analysis were homozygous and contained a single copy of the transgene. METHODS: Each transgenic soybean seed was screened for transgene presence and recombinant protein expression via PCR and western blotting.  Whole seed mRNA was extracted and cDNA libraries constructed for Illumina sequencing.  Following alignment to the soybean reference genome, differential gene expression analysis was conducted using edgeR and cufflinks.  Functional analysis of differentially expressed genes was carried out using the gene ontology analysis tool AgriGO. RESULTS: The transcriptomes of nine seeds from each transgenic line were sequenced and compared with wild type seeds. Native soybean gene expression was significantly altered in line 764 (mSEB) with more than 3000 genes being upregulated or downregulated. ST77 (hTG) and ST111 (hMBP) had significantly less differences with 52 and 307 differentially expressed genes respectively. Gene ontology enrichment analysis found that the upregulated genes in the 764 line were annotated with functions related to endopeptidase inhibitors and protein synthesis, but suppressed expression of genes annotated to the nuclear pore and to protein transport. No significant gene ontology terms were detected in ST77, and only a few genes involved in photosynthesis and thylakoid functions were downregulated in ST111. Despite these differences, transgenic plants and seeds appeared phenotypically similar to non-transgenic controls. There was no correlation between recombinant protein expression level and the quantity of differentially expressed genes detected. CONCLUSIONS: Measurable unscripted gene expression changes were detected in the seed transcriptomes of all three transgenic soybean lines analyzed, with line 764 being substantially altered. Differences detected at the transcript level may be due to T-DNA insert locations, random mutations following transformation or direct effects of the recombinant protein itself, or a combination of these. The physiological consequences of such changes remain unknown.

Soybean seeds: a practical host for the production of functional subunit vaccines.

Soybean seeds possess several inherent qualities that make them an ideal host for the production of biopharmaceuticals when compared with other plant-based and non-plant-based recombinant expression systems (e.g., low cost of production, high protein to biomass ratio, long-term stability of seed proteins under ambient conditions, etc.). To demonstrate the practicality and feasibility of this platform for the production of subunit vaccines, we chose to express and characterize a nontoxic form of S. aureus enterotoxin B (mSEB) as a model vaccine candidate. We show that soy-mSEB was produced at a high vaccine to biomass ratio and represented ~76 theoretical doses of human vaccine per single soybean seed. We localized the model vaccine candidate both intracellularly and extracellularly and found no difference in mSEB protein stability or accumulation relative to subcellular environment. We also show that the model vaccine was biochemically and immunologically similar to native and recombinant forms of the protein produced in a bacterial expression system. Immunization of mice with seed extracts containing mSEB mounted a significant immune response within 14 days of the first injection. Taken together, our results highlight the practicality of soybean seeds as a potential platform for the production of functional subunit vaccines.

Sublethal staphylococcal enterotoxin B challenge model in pigs to evaluate protection following immunization with a soybean-derived vaccine.

In an effort to develop a sustainable platform for manufacturing protein-based vaccine candidates, we expressed a triple mutant of staphylococcal enterotoxin B carrying the L45R, Y89A, and Y94A modifications in transgenic soybean seeds (soy-mSEB). Soy-mSEB possessed no detectable superantigen activity in vitro. We found that this soybean-derived, nontoxic mutant of SEB could be stably expressed, stored in seeds for extended periods at room temperature without degradation, and easily purified from contaminating soy proteins. Vaccination of pigs with purified soy-mSEB, or the identical triple mutant expressed in Escherichia coli (E. coli-mSEB), resulted in high antibody titers against the native toxin in immunized animals. In fact, titers were indistinguishable regardless of the immunogen used, demonstrating the equivalence of soy-mSEB and E. coli-mSEB vaccinations. Antisera from either immunized group were able to block native SEB superantigen activity in an in vitro neutralization assay. Similar results were obtained when immunized animals were challenged with a sublethal dose of native toxin. Significant reductions in toxin-induced serum cytokine levels were observed in soy-mSEB- and E. coli-mSEB-immunized pigs compared to control animals. The reductions in SEB-induced cytokine responses were similar regardless of the immunogen used for vaccination. Surprisingly, however, some clinical symptoms, such as prostration, lethargy, emesis, and/or diarrhea, were still observed in all immunized animals. These studies demonstrate the potential for soybean-derived proteins as a platform technology for sustainable vaccine manufacturing and the usefulness of a sublethal challenge model in pigs for evaluating the efficacy of potential SEB vaccine candidates.

Recombinant expression of homodimeric 660 kDa human thyroglobulin in soybean seeds: an alternative source of human thyroglobulin.

Soybean seeds possess many qualities that make them ideal targets for the production of recombinant proteins. However, one quality often overlooked is their ability to stockpile large amounts of complex storage proteins. Because of this characteristic, we hypothesized that soybean seeds would support recombinant expression of large and complex proteins that are currently difficult or impossible to express using traditional plant and non-plant-based host systems. To test this hypothesis, we transformed soybeans with a synthetic gene encoding human thyroglobulin (hTG)-a 660 kDa homodimeric protein that is widely used in the diagnostic industry for screening and detection of thyroid disease. In the absence of a recombinant system that can produce recombinant hTG, research and diagnostic grade hTG continues to be purified from cadaver and surgically removed thyroid tissue. These less-than-ideal tissue sources lack uniform glycosylation and iodination and therefore introduce variability when purified hTG is used in sensitive ELISA screens. In this study, we report the successful expression of recombinant hTG in soybean seeds. Authenticity of the soy-derived protein was demonstrated using commercial ELISA kits developed specifically for the detection of hTG in patient sera. Western analyses and gel filtration chromatography demonstrated that recombinant hTG and thyroid-purified hTG are biologically similar with respect to size, mass, charge and subunit interaction. The recombinant protein was stable over three generations and accumulated to ~1.5% of total soluble seed protein. These results support our hypothesis that soybeans represent a practical alternative to traditional host systems for the expression of large and complex proteins.

Oral vaccine formulations stimulate mucosal and systemic antibody responses against staphylococcal enterotoxin B in a piglet model.

Despite the potential for its use as an agent of biowarfare or bioterrorism, no approved vaccine against staphylococcal enterotoxin B (SEB) exists. Nontoxic, mutant forms of SEB have been developed; however, it has been difficult to determine the efficacy of such subunit vaccine candidates due to the lack of superantigen activity of native SEB in rodents and due to the limitations of primate models. Since pigs respond to SEB in a manner similar to that of human subjects, we utilized this relevant animal model to investigate the safety and immunogenicity of a triple mutant of SEB carrying the amino acid changes L45R, Y89A, and Y94A. This recombinant mutant SEB (rmSEB) did not possess superantigen activity in pig lymphocyte cultures. Furthermore, rmSEB was unable to compete with native SEB for binding to pig leukocytes. These in vitro studies suggested that rmSEB could be a safe subunit vaccine. To test this possibility, piglets immunized orally with rmSEB formulations experienced no significant decrease in food consumption and no weight loss during the vaccination regimen. Oral vaccination with 1-mg doses of rmSEB on days 0, 7, 14, and 24 resulted in serum IgG and fecal IgA levels by day 36 that cross-reacted with native SEB. Surprisingly, the inclusion of cholera toxin adjuvant in vaccine formulations containing rmSEB did not result in increased antibody responses compared to formulations using the immunogen alone. Taken together, these studies provide additional evidence for the potential use of nontoxic forms of SEB as vaccines.

Chloroplast targeting of FanC, the major antigenic subunit of Escherichia coli K99 fimbriae, in transgenic soybean.

Enterotoxigenic Escherichia coli (ETEC) strains are a major cause of enteric diseases affecting livestock and humans. Edible transgenic plants producing E. coli fimbrial subunit proteins have the potential to vaccinate against these diseases, but have not reached their full potential as a renewable source of oral vaccines due in part to insufficient levels of recombinant protein accumulation. Previously, we reported that cytosol targeting of the E. coli K99 fimbrial subunit antigen resulted in FanC accumulation to approximately 0.4% of total soluble protein in soybean leaves (Piller et al. in Planta 222:6-18, 2005). In this study, we report on the subcellular targeting of FanC to chloroplasts. Twenty-two transgenic T1 progeny derived from seven individual T0 transformation events were characterized, and 17 accumulated transgenic FanC. All of the characterized events displayed relatively low T-DNA complexity, and all exhibited proper targeting of FanC to the chloroplast. Accumulation of chloroplast-targeted FanC was approximately 0.08% of total soluble leaf protein, or approximately 5-fold less than cytosol-targeted FanC. Protein analysis of leaves at various stages of maturity suggested stability of chloroplast-targeted FanC throughout leaf maturation. Furthermore, mice immunized intraperitoneally with protein extract derived from transgenic leaves expressing chloroplast-targeted FanC developed significant antibody titers against FanC. This is the first report of subcellular targeting of a vaccine subunit antigen in soybean.

Expression and immunogenicity of an Escherichia coli K99 fimbriae subunit antigen in soybean.

Enterotoxigenic Escherichia coli (ETEC) cause acute diarrhea in humans and farm animals, and can be fatal if the host is left untreated. As a potential alternative to traditional needle vaccination of cattle, we investigated the feasibility of expressing the major K99 fimbrial subunit, FanC, in soybean (Glycine max) for use as an edible subunit vaccine. As a first step in this developmental process, a synthetic version of fanC was optimized for expression in the cytosol and transferred to soybean via Agrobacterium-mediated transformation. Western analysis of T(0) events revealed the presence of a peptide with the expected mobility for FanC in transgenic protein extracts, and immunofluorescense confirmed localization to the cytosol. Two T(0) lines, which accumulated FanC to levels near 0.5% of total soluble protein, were chosen for further molecular characterization in the T(1) and T(2) generations. Mice immunized intraperitoneally with protein extract derived from transgenic leaves expressing synthetic FanC developed significant antibody titers against bacterially derived FanC and produced antigen-specific CD4(+) T lymphocytes, demonstrating the ability of transgenic FanC to function as an immunogen. These experiments are the first to demonstrate the expression and immunogenicity of a model subunit antigen in the soybean system, and mark the first steps toward the development of a K99 edible vaccine to protect against ETEC.