Celine A. Hayden

Manipulating corn germplasm to increase recombinant protein accumulation.

Using plants as biofactories for industrial enzymes is a developing technology. The application of this technology to plant biomass conversion for biofuels and biobased products has potential for significantly lowering the cost of these products because of lower enzyme production costs. However, the concentration of the enzymes in plant tissue must be high to realize this goal. We describe the enhancement of the accumulation of cellulases in transgenic maize seed as a part of the process to lower the cost of these dominant enzymes for the bioconversion process. We have used breeding to move these genes into elite and high oil germplasm to enhance protein accumulation in grain. We have also explored processing of the grain to isolate the germ, which preferentially contains the enzymes, to further enhance recovery of enzyme on a dry weight basis of raw materials. The enzymes are active on microcrystalline cellulose to release glucose and cellobiose.

Bioencapsulation of the hepatitis B surface antigen and its use as an effective oral immunogen.

Hepatitis B remains a major global health problem despite the availability of a safe and effective vaccine. Segments of the population lack access to or respond poorly to the parenteral vaccine, perpetuating the infection-transmission cycle. A low cost, orally delivered vaccine has the potential to alleviate many of these problems. Here we describe the expression of a bioencapsulated hepatitis B surface antigen (HBsAg) in maize and its immunogenicity, demonstrating for the first time a commercially feasible oral subunit vaccine production system for a major disease. This work surmounts previous barriers to plant-produced vaccines by expressing HBsAg at much higher levels and retaining antigen immunogenicity post-processing: factors which facilitated a robust immune response in mice without the need for an adjuvant. This method provides a practical solution to the delivery of a low-cost, stable oral vaccine.

Translational regulation of Arabidopsis XIPOTL1 is modulated by phosphocholine levels via the phylogenetically conserved upstream open reading frame 30

In Arabidopsis thaliana, XIPOTL1 encodes a phosphoethanolamine N-methyltransferase with a central role in phosphatidylcholine biosynthesis via the methylation pathway. To gain further insights into the mechanisms that regulate XIPOTL1 expression, the effect of upstream open reading frame 30 (uORF30) on the translation of the major ORF (mORF) in the presence or absence of endogenous choline (Cho) or phosphocholine (PCho) was analysed in Arabidopsis seedlings. Dose-response assays with Cho or PCho revealed that both metabolites at physiological concentrations are able to induce the translational repression of a mORF located downstream of the intact uORF30, without significantly altering its mRNA levels. PCho profiles showed a correlation between increased endogenous PCho levels and translation efficiency of a uORF30-containing mORF, while no correlation was detectable with Cho levels. Enhanced expression of a uORF30-containing mORF and decreased PCho levels were observed in the xipotl1 mutant background relative to wild type, suggesting that PCho is the true mediator of uORF30-driven translational repression. In Arabidopsis, endogenous PCho content increases during plant development and affects root meristem size, cell division, and cell elongation. Because XIPOTL1 is preferentially expressed in Arabidopsis root tips, higher PCho levels are found in roots than shoots, and there is a higher sensitivity of this tissue to translational uORF30-mediated control, it is proposed that root tips are the main site for PCho biosynthesis in Arabidopsis.

Production of highly concentrated, heat-stable hepatitis B surface antigen in maize

Plant-based oral vaccines are a promising emergent technology that could help alleviate disease burden worldwide by providing a low-cost, heat-stable, oral alternative to parenterally administered commercial vaccines. Here, we describe high-level accumulation of the hepatitis B surface antigen (HBsAg) at a mean concentration of 0.51%TSP in maize T1 seeds using an improved version of the globulin1 promoter. This concentration is more than fourfold higher than any previously reported lines. HBsAg expressed in maize seeds was extremely heat stable, tolerating temperatures up to 55 °C for 1 month without degradation. Optimal heat stability was achieved after oil extraction of ground maize material, either by supercritical fluid extraction or hexane treatment. The contributions of this material towards the development of a practical oral vaccine delivery system are discussed.

Oral delivery of wafers made from HBsAg-expressing maize germ induces long-term immunological systemic and mucosal responses

BACKGROUND The hepatitis B surface antigen (HBsAg) has been administered over the last 20 years as a parenteral vaccine against the hepatitis B virus (HBV). Despite high seroconversion rates, chronic infection rates are still high worldwide. Orally delivered vaccines provide a practical alternative to injected vaccines, potentially helping poorly responding populations and providing a viable alternative for populations in remote locations. Anamnestic responses are vital to establishing the efficacy of a given vaccine and have been assessed in this study using a plant-based oral delivery platform expressing HBsAg. METHODS Long-term immunological memory was assessed in mice injected with a primary dose of Recombivax and boosted with orally-delivered HBsAg wafers, control wafers, or parenterally-delivered commercial vaccine (Recombivax). RESULTS Mice boosted with HBsAg orally-administered wafers displayed sharp increases in mucosal IgA titers in fecal material and steep increases in serum IgA, whereas mice boosted with Recombivax showed no detectable levels of IgA in either fecal or serum samples following four boosting treatments. Long-term memory in the orally-treated mice was evidenced by sustained fecal IgA, and serum IgA, IgG, and mIU/mL over one year, while Recombivax-treated mice displayed sustained serum IgG and mIU/mL. Furthermore, sharp increases in these same antibodies were induced after re-boosting at 47 and 50 weeks post-primary injection. CONCLUSIONS Orally-delivered vaccines can provide long-term immune responses mucosally and systemically. For sexually-transmitted diseases that can be acquired at mucosal surfaces, such as HBV, an oral delivery platform may provide added protection over a conventional parenterally administered vaccine.

Supercritical fluid extraction provides an enhancement to the immune response for orally-delivered hepatitis B surface antigen.

The hepatitis B virus continues to be a major pathogen worldwide despite the availability of an effective parenteral vaccine for over 20 years. Orally-delivered subunit vaccines produced in maize may help to alleviate the disease burden by providing a low-cost, heat-stable alternative to the parenteral vaccine. Oral subunit vaccination has been an elusive goal due to the large amounts of antigen required to induce an immunologic response when administered through the digestive tract. Here we show that high levels of HBsAg were obtained in maize grain, the grain was formed into edible wafers, and wafers were fed to mice at a concentration of approximately 300 μg/g. When these wafers were made with supercritical fluid extraction (SFE)-treated maize material, robust IgG and IgA responses in sera were observed that were comparable to the injected commercial vaccine (Recombivax(®)). In addition, all mice administered SFE wafers showed high secretory IgA titers in fecal material whereas Recombivax(®) treated mice showed no detectable titer. Increased salivary IgA titers were also detected in SFE-fed mice but not in Recombivax(®) treated mice. Wafers made from hexane-treated or full fat maize material induced immunologic responses, but fecal titers were attenuated relative to those produced by SFE-treated wafers. These responses demonstrate the feasibility of using a two-dose oral vaccine booster in the absence of an adjuvant to induce immunologic responses in both sera and at mucosal surfaces, and highlight the potential limitations of using an exclusively parenteral dosing regime.

Biochemical and biophysical characterization of maize-derived HBsAg for the development of an oral vaccine.

Although a vaccine against hepatitis B virus (HBV) has been available since 1982, it is estimated that 600,000 people die every year due to HBV. An affordable oral vaccine could help alleviate the disease burden and to this end the hepatitis B surface antigen (HBsAg) was expressed in maize. Orally delivered maize material induced the strongest immune response in mice when lipid was extracted by CO2 supercritical fluid extraction (SFE), compared to full fat and hexane-extracted material. The present study provides a biochemical and biophysical basis for these immunological differences by comparing the active ingredient in the differently treated maize material. Purified maize-derived HBsAg underwent biophysical characterization by gel filtration, transmission electron microscopy (TEM), dynamic light scattering (DLS), UV-CD, and fluorescence. Gel filtration showed that HBsAg forms higher-order oligomers and TEM demonstrated virus-like particle (VLP) formation. The VLPs obtained from SFE were more regular in shape and size compared to hexane or full fat material. In addition, SFE-derived HBsAg showed the greatest extent of α-helical structure by far UV-CD spectrum. Fluorescence experiments also revealed differences in protein conformation. This work establishes SFE-treated maize material as a viable oral vaccine candidate and advances the development of the first oral subunit vaccine.

Synergistic Activity of Plant Extracts with Microbial Cellulases for the Release of Free Sugars

Agricultural lignocellulosic waste such as corn stover is a potential source of inexpensive, abundant, and renewable biomass for the production of bioethanol. The enzymatic process for the economically viable breakdown of cellulose to ethanol relies on the availability of inexpensive microbial cellulases. Although the cost of cellulase has decreased in recent years, current costs still preclude the production of economically viable bioethanol from lignocellulose. Substantive efforts in this lab are being directed to transgenic production of cellulases in maize in order to boost efficiency both of production of enzymes and degradation of corn stover. We serendipitously observed that the addition of non-transgenic maize seed extracts to cellulose and microbial enzymes potentiated free sugar release by as much as 20-fold. Further, this synergistic effect between cellulase enzymes and extract was seen with a variety of plant species and tissue extracts, but varied in efficiency, and was optimal at low concentrations of cellulases. Although the nature of the synergistic molecule is not known, the use of extracts to potentiate cellulose breakdown provides opportunities for a clearer mechanistic understanding of the degradation process as well as an economical way to improve the efficiency of cellulases to produce more cost-effective bioethanol from agricultural waste.

An Oral Vaccine for Hepatitis B: Challenges, Setbacks, and Breakthroughs

Despite the commercialization of a highly effective injectable vaccine for over 20 years, the hepatitis B virus (HBV) continues to be a significant health concern worldwide. An estimated 240 million people are chronically infected with the virus despite efforts by the World Health Organization (WHO) to implement child vaccination programs in all WHO member countries for over 10 years. Plants hold the promise of bringing us one step closer to eradication of the disease by providing a low-cost, heat stable, oral alternative to the parenteral vaccine. Several technical hurdles have been overcome over the last several years and a maize-based vaccine has emerged as the front-runner oral vaccine candidate. This review outlines those technical hurdles in the context of hepatitis B epidemiology and socioeconomic factors influencing adoption of vaccination compliance. Additional hurdles to commercialization, such as regulatory considerations and production, are also discussed.