Tackaberry, ES; Dudani, AK; Prior, F; Tocchi, M; Sardana, R; Altosaar, I; Ganz, PR

Vaccine, vol. 17, no. 23-24, pp. 3020-3029, 6 Aug 1999

Plant seeds offer unique opportunities for the production and delivery of oral subunit vaccines. We have used the immunodominant glycoprotein B complex of human cytomegalovirus (HCMV), introduced into tobacco plants, as a model system for studying the merit of this promising approach. Given the advantages of expressing proteins in seeds, a novel expression vector was developed incorporating regulatory sequences of glutelin, the major rice seed storage protein, to direct synthesis of recombinant glycoprotein B. Analysis of genomic DNA of 28 selected tobacco transformants by PCR amplification showed that 71% harboured the gB cDNA, a finding further documented by Southern blotting. Specific immunoassays of protein extracts from seeds of positive plants showed that all were producing antigenic glycoprotein B at levels ranging from 70-146 ng/mg extracted protein. In addition, similarity with native glycoprotein B produced in HCMV-infected cells was also demonstrated by inhibition of immunofluorescence on HCMV-infected human fibroblasts. These data are the first to report the expression of an immunodominant antigen of HCMV in plant tissues, indicating the fidelity with which this very large heterologous viral glycoprotein can be synthesized in this model system.

Russell, DA

Current Topics in Microbiology and Immunology [Curr. Top. Microbiol. Immunol.], vol. 240, pp. 119-138, 1999

In the 24 years since the initial publication describing monoclonal antibodies field has advanced in the manipulation, production, and use of such biologics. While the molecular cloning of the antibody coding regions and advances in immunology have helped direct the engineering of antibodies, it has also paved the way for alternate expression systems. Given the efficiency of agricultural production systems, and successes in commercialization of transgenic crops, it is not surprising that plants have been investigated for such production. The use of plants to replace more traditional mammalian cell production is based on both systems having similar protein synthesis machinery, including many posttranslational processing steps. However, for commercial production of antibodies to the standards necessary for therapeutic use, any new system must be accurate, reproducible, and competitive. This chapter discusses the potential of therapeutic antibody production in transgenic plants to meet those criteria. The concepts described have been applied to the first human clinical trial for an injectable therapeutic from transgenic plants.

Richter, L; Kipp, PB

Current Topics in Microbiology and Immunology [Curr. Top. Microbiol. Immunol.], vol. 240, pp. 159-176, 1999

Vaccinations are among the more cost-effective health care procedures. In the United States and Europe, a majority of newborn children are vaccinated against ten diseases (Recommended childhood immunization schedule from Center for Disease Control, USA). Recently in the United States hepatitis B was added to the recommended vaccinations for infants. Of these different immunizations, only one is an oral vaccine with the others requiring injections. In addition, approximately thirty new vaccines are currently under development in the United States. In contrast to Western countries, vaccines are in limited use in many developing countries, with children receiving the "Expanded Program on Immunization" (EPI) vaccines against six diseases. These are recommended, and in many countries financed, by the World Health Organization. The cost of vaccines is one factor preventing further use of vaccination, leaving hundreds of thousands of children susceptible to preventable diseases. The principle costs of most commercial vaccines are production, packaging and delivery. Injectable vaccines incur further expenses related to the use and disposal of needles and syringes, trained personnel to administer injections, and refrigeration required during shipping and storage. These same economic factors prevent widespread vaccination of livestock, poultry and swine against preventable diseases. The Children's Vaccine Initiative called for new technologies to make vaccines more widely available. This includes low cost production systems and to further develop oral vaccines. Oral vaccines are desirable due to their ease of administration and patients acceptance of noninjected vaccinations. Another advantage is that oral vaccines may stimulate production of mucosal antibodies more effectively than injected vaccines. This is important as the mucosal immune system is a first line of defense against many disease organisms. Vaccines are designed to elicit an immune response without causing disease. Typical vaccines are composed of killed or attenuated disease-causing organisms. Recombinant subunit vaccines are desirable as an alternative with potentially fewer side effects than delivering the whole organism. Recombinant subunit vaccines do not contain an infectious agent, and thus are safer to administer and prepare, and doses are more uniform. Advances in molecular biology of diseases have identified many candidate proteins or peptides that may function as effective subunit vaccines. Recombinant vaccines have potential for being highly effective in preventing disease, both in humans and animals, but are rather costly to produce, and therefore are in limited use worldwide. The choice of which system to use to produce a recombinant vaccine must take into consideration their advantages and disadvantages, costs of production, and the amount of product required on a global scale. For some vaccine antigens, transgenic plants may provide an ideal expression system, in which transgenic plant material can be fed directly to subjects as their oral dose of recombinant vaccine.

Zeitlin, L; Olmsted, SS; Moench, TR; Co, MS; Martinell, BJ; Paradkar, VM; Russell, DR; Queen, C; Cone, RA; Whaley, KJ*

Nature Biotechnology [Nat. Biotechnol.], vol. 16, no. 13, pp. 1361-1364, Dec 1998

The ability to produce monoclonal antibodies (Mabs) in plants offers the opportunity for the development of an inexpensive method of mucosal immunoprotection against sexually transmitted diseases. To investigate the suitability of plant-expressed Mabs for vaginal preventive applications, we compared a humanized anti-herpes simplex virus 2 (HSV-2) Mab expressed in mammalian cell culture with the same antibody expressed in soybean. We found these Mabs to be similar in their stability in human semen and cervical mucus over 24 h, their ability to diffuse in human cervical mucus, and their efficacy for prevention of vaginal HSV-2 infection in the mouse.

Tacket, CO; Mason, HS; Losonsky, G; Clements, JD; Levine, MM; Arntzen, CJ

Nature Medicine [Nat. Med.], vol. 4, no. 5, pp. 607-609, May 1998

Compared with vaccine delivery by injection, oral vaccines offer the hope of more convenient immunization strategies and a more practical means of implementing universal vaccination programs throughout the world. Oral vaccines act by stimulating the immune system at effector sites (lymphoid tissue) located in the gut. Genetic engineering has been used with variable success to design living and non-living systems as a means to deliver antigens to these sites and to stimulate a desired immune response. More recently, plant biotechnology techniques have been used to create plants which contain a gene derived from a human pathogen; the resultant plant tissues will accumulate an antigenic protein encoded by the foreign DNA. In pre-clinical trials, we found that antigenic proteins produced in transgenic plants retained immunogenic properties when purified; if injected into mice the antigen caused production of protein-specific antibodies. Moreover, in some experiments, if the plant tissues were simply fed to mice, a mucosal immune response occurred. The present study was conducted as a proof of principle to determine if humans would also develop a serum and/or mucosal immune response to an antigen delivered in an uncooked foodstuff.

Arakawa, T; Chong, DKX; Langridge, WHR*

Nature Biotechnology [Nat. Biotechnol.], vol. 16, no. 3, pp. 292-297, Mar 1998

Transgenic potatoes were engineered to synthesize a cholera toxin B subunit (CTB) pentamer with affinity for G sub(M1)-ganglioside. Both serum and intestinal CTB-specific antibodies were induced in orally immunized mice. Mucosal antibody titers declined gradually after the last immunization but were restored following an oral booster of transgenic potato. The cytopathic effect of cholera holotoxin (CT) on Vero cells was neutralized by serum from mice immunized with transgenic potato tissues. Following intraileal injection with CT, the plant-immunized mice showed up to a 60% reduction in diarrheal fluid accumulation in the small intestine. Protection against CT was based on inhibition of enterotoxin binding to the cell-surface receptor G sub(M1)-ganglioside. These results demonstrate the ability of transgenic food plants to generate protective immunity in mice against a bacterial enterotoxin.

Palmgren, G

Trends in Genetics [TRENDS GENET.], vol. 13, no. 9, p.348, Sep 1997

There was good news and bad news at this meeting. The good news was that we now have such extensive knowledge of the genes involved in many biosynthetic pathways that transgenic plants could well become the 'green' environmentally safe factories of the future. The bad news was that even now, 14 years after it first became possible to transform plants, the technology is still too slow, inefficient and unpredictable. One session at the symposium was devoted to gene suppression (or cosuppression), a problem facing most scientists working with transgenic plants. Joseph Mol (Free University, Amsterdam, The Netherlands) reported that transformation of petunia with the chalcone synthase A gene in several cases leads to suppression of both the transgene(s) and the endogenous gene. Suppression is accompanied by extensive DNA methylation, which is now believed to result in premature termination of transcription. Consequently, aberrant RNA species are formed that trigger sequence-specific RNA degradation either directly or indirectly through incorrect splicing, transport and so on. A number of researchers have used genetically modified plant viruses to express novel proteins in untransformed plants. Guy Della-Cioppa (Biosource Technologies Inc., Vacaville, CA, USA) explained that after successful field trials of tobacco infected with modified tobacco mosaic virus expressing genes encoding, for example, green fluorescent protein, the company has gained official approval and expects to start commercial production of pharmaceuticals this year. One of the most exciting technical advances was presented by Ramesh Kumar (Kimeragen Inc., Newtown, PA, USA) -- an in vivo site-directed mutagenesis approach whereby only very few nucleotides are substituted by homologous recombination. Using a chimeric, single-stranded RNA-DNA molecule, the method exploits the high efficiency of RNA--DNA duplex formation. The synthetic molecule contains a mutated sequence of the gene of interest, and recombines with the endogenous gene after protoplast transfection. After cell division, the mutation is stably integrated and plants can be regenerated. This method seems promising, for example, for making gene knockouts by simply introducing stop codons in otherwise unaltered plants. However, results presented by John Shanklin (Brookhaven Natl Lab., New York, USA) opened up further applications. By comparing crystal structure and sequence data for various fatty acid synthases, a small number of amino acids were identified that are essential for chain length specificity and double-bond position specificity. Changing these amino acids leads to the controlled alteration of the fatty acid synthase specificity. In addition, changing seven amino acids was sufficient to convert a desaturase into a hydroxylase. Hydroxylated fatty acids produced this way in transgenic Arabidopsis were shown to be included into the fatty acid metabolism of the plant and to be further elongated or desaturated. This creates the potential to make products based on hydroxy fatty acids, such as paint, nylon, resin and lubricants in transgenic plants. In accordance with this, Toni Kutchan (Univ. of Munich, Germany) illustrated how novel alkaloids could be synthesized in vitro from enzymes fed with extrinsic substrates, and she anticipated that more knowledge of such enzymatic 'side effects' and substrate shuffling would lead to production of completely new alkaloids in transgenic plants.

McGarvey, PB; Hammond, J; Dienell, MM; Hooper, DC; Fu, ZF; Dietzschold, B; Koprowski, H; Michaels, FH

BIO/TECHNOLOGY, vol. 13, no. 13, pp. 1484-1487, 1995

We have engineered tomato plants (Lycopersicon esculentum Mill var. UC82b) to express a gene for the glycoprotein (G-protein), which coats the outer surface the rabies virus. The recombinant constructs contained the G-protein gene from the ERA strain of rabies virus, including the signal peptide, under the control of the 35S promoter of cauliflower mosaic virus. Plants were transformed by Agrobacterium tumefaciens-mediated transformation of cotyledons and tissue culture on selective media. PCR confirmed the presence of the G-protein gene in plants surviving selection. Northern blot analysis indicated that RNA of the appropriate molecular weight was produced in both leaves and fruit of the transgenic plants. The recombinant G-protein was immunoprecipitated and detected by Western blot from leaves and fruit using different antisera. The G-protein expressed in tomato appeared as two distinct bands with apparent molecular mass of 62 and 60 kDa as compared to the 66 kDa observed for G-protein from virus grown in BHK cells. Electron microscopy of leaf tissue using immunogold-labeling and antisera specific for rabies G-protein showed localization of the G-protein to the Golgi bodies, vesicles, plasmalemma and cell walls of vascular parenchyma cells. In light of our previous demonstration that orally administered rabies G-protein from the same ERA strain elicits protective immunity in animals, these transgenic plants should provide a valuable tool for the development of edible oral vaccines.

Mason, HS; Arntzen, CJ

Trends in Biotechnology [TRENDS BIOTECHNOL.], vol. 13, no. 9, pp. 388-392, 1995

Transgenic plants that express foreign proteins with industrial or pharmaceutical value represent an economical alternative to fermentation-based production systems. Specific vaccines have been produced in plants as a result of the transient or stable expression of foreign genes. It has recently been shown that genes encoding antigens of bacterial and viral pathogens can be expressed in plants in a form in which they retain native immunogenic properties. Transgenic potato tubers expressing a bacterial antigen stimulated humoral and mucosal immune responses when they were provided as food. These results provide 'proof of concept' for the use of plants as a vehicle to produce vaccines.

Mason, HS; Lam, DMan-Kit; Arntzen, CJ

Proceedings of the National Academy of Sciences, USA [PROC. NATL. ACAD. SCI. USA.], vol. 89, no. 24, pp. 11745-11749, 1992

Tobacco plants were genetically transformed with the gene encoding hepatitis B surface antigen (HBsAg) linked to a nominally constitutive promoter. Enzyme-linked immunoassays using a monoclonal antibody directed against human serum-derived HBsAg revealed the presence of HBsAg in extracts of transformed leaves at levels that correlated with mRNA abundance. This suggests that there were no major inherent limitations of transcription or translation of this foreign gene in plants. Recombinant HBsAg was purified from transgenic plants by immunoaffinity chromatography and examined by electron microscopy. Because the HBsAg produced in transgenic plants is antigenically and physically similar to the HBsAg particles derived from human serum and recombinant yeast, which are used as vaccines, we conclude that transgenic plants hold promise as low-cost vaccine production systems.

Kapusta, J.; Modelska, A.; Figlerowicz, M.; Pniewski, T.; Letellier, M.; Lisowa, O.; Yusibov, V.; Koprowski, H.; Plucienniczak, A.; Legocki, A.B.

FASEB Journal [FASEB J.], vol. 13, no. 13, p. 1796-1799, Oct 1999

The infectious hepatitis B virus represents 42 nm spherical double-shelled particles. However, analysis of blood from hepatitis B virus carriers revealed the presence of smaller 22 nm particles consisting of a viral envelope surface protein. These particles are highly immunogenic and have been used in the design of hepatitis B virus vaccine produced in yeast. Upon expression in yeast, these proteins form virus-like particles that are used for parenteral immunization. Therefore, the DNA fragment encoding hepatitis B virus surface antigen was introduced into Agrobacterium tumerifacience LBA4404 and used to obtain transgenic lupin (Lupinus luteus L.) and lettuce (Lactuca sativa L.) cv. Burpee Bibb expressing envelope surface protein. Mice that were fed the transgenic lupin tissue developed significant levels of hepatitis B virus-specific antibodies. Human volunteers, fed with transgenic lettuce plants expressing hepatitis B virus surface antigen, developed specific serum-IgG response to plant produced protein.