INFORMATION SYSTEMS FOR BIOTECHNOLOGY

March 2002

COVERING AGRICULTURAL AND ENVIRONMENTAL BIOTECHNOLOGY DEVELOPMENTS

---

.pdf version

IN THIS ISSUE:
NRC Report Suggests Improvements to Federal Oversight of Transgenic Plants
Report of Transgenes in Mexican Corn Called into Question
Joint Statement on the Mexican GM Maize Scandal
Joint Statement in Support of Scientific Discourse in Mexican GM Maize Scandal
Bt Corn Poses "No Significant Risk" to Monarchs
Edible Foot and Mouth Vaccination from Transgenic Plants
Mammalian Cells Express Spider Silk Proteins
Inventors Hash Out Priority for Cloning Technology and Other Biotech Law News
Upcoming Meetings

NRC REPORT SUGGESTS IMPROVEMENTS TO FEDERAL OVERSIGHT OF TRANSGENIC PLANTS

The environmental effects of transgenic plants and the adequacy of federal oversight were evaluated in a landmark report released February 21 by the National Research Council (NRC).¹ The report, commissioned January 2000 by the Animal and Plant Health Inspection Service (APHIS) of the US Department of Agriculture, evaluated underlying scientific premises and assumptions, scope, and adequacy of environmental reviews, as well as approaches to environmental monitoring of effects of transgenic plants. The report was authored by a committee of twelve leading university scientists with expertise in plant sciences, molecular genetics, insect ecology, biotechnology, and other relevant areas. Committee chair Fred Gould of North Carolina State University summarized the report's findings,² saying that the USDA "has substantially improved its regulation of transgenic plants, but the process could be improved further by soliciting greater public input, enhancing scientific peer review, and more clearly presenting the data and methods behind regulatory decisions. Our report provides a detailed road map for the federal government to follow as it reinforces its assessment of environmental risks."

Key findings of the NRC report
The committee felt a need to place potential impacts of transgenic crops within the context of environmental effects of other agricultural practices and technologies. They did not identify any strict distinctions between the types of environmental risks posed by plants produced through genetic engineering and those posed by conventional breeding. The committee found that it should be possible to quickly screen genetically modified plants for potential environmental risk and then to conduct detailed tests only on those plants where preliminary screening indicates potential risk.

Field testing of most transgenic plants goes forward within the "notification" process in which applicants notify APHIS that the plant meets general guidelines for not causing environmental impacts. If APHIS agrees, the applicant can proceed with the field test. There is no public or independent scientific input into the decision-making process. The committee found the notification process conceptually appropriate, but felt that there was a need to reexamine which transgenic plants should be tested and commercialized through the notification process. More detailed investigations should be conducted when preliminary testing indicates possible hazards.

Most biotech companies commercialize transgenic plants by petitioning for non-regulated status. Under current procedure, APHIS conducts an environmental review, publishes it in the Federal Register, and allows 60 days for comments. However, the committee felt that the process should be made significantly more transparent and rigorous by enhanced scientific peer review and use of more explicit presentation of methods, data, analyses, and interpretations. The committee felt that before reaching precedent-setting decisions regarding field testing or deregulation, APHIS should solicit broad scientific and public review using means beyond the Federal Register, and should convene a scientific advisory group before making changes in regulatory policy. The committee also recommended that in the future, APHIS should include any potential impacts of transgenic plants on regional farming practices or systems in its deregulation assessments.

The committee found APHIS's assessment of hazards involving insect pests acquiring resistance to toxins expressed by transgenes and impacts on non-target species superficial. They recommended that the agency should either increase the rigor of its assessments or defer to the US Environmental Protection Agency in such cases.

Once a product is deregulated, APHIS neither conducts post-commercialization monitoring, nor requires companies to do so. The committee recommended post-commercialization monitoring to validate pre-commercialization testing and detect any unanticipated or long-term environmental impacts.

Reactions to the NRC report
In a statement released upon the NRC report publication, APHIS Administrator Bobby Acord³ noted that the NRC recommendations were provided "as a means to help improve an already functioning system." He noted that USDA has already addressed some of the issues raised in the report. APHIS is working to incorporate independent scientific input into the notification process for field trials of transgenic plants. The agency is considering how best to encourage public comment and receive broader scientific input when considering permits for movement, importation, and field testing of genetically modified plants. APHIS also is assessing various options for monitoring transgenic plant products that already have been commercialized, and for reregulating products if a plant pest risk is discovered.

Environmentalists and groups critical of genetically modified foods regarded the report as support for their view that regulatory oversight has been inadequate. Margaret Mellon of the Union of Concerned Scientists told the New York Times⁴ that, "The real question raised by this report is whether a rickety system that hasn't been very rigorous but probably has served well enough to date is adequate enough to take us into the future." The answer delivered by the panel, she said, is that the system is not up to the task. In a statement quoted by Reuters,⁵ Genetically Engineered Food Alert said, "The report is justifiably critical of (USDA's) cult of secrecy, shutting out the public from reviewing the agency's decisions."

Representatives of the biotechnology industry tended to emphasize the degree to which the NRC committee supported the existing regulatory system. Michael J. Philips,⁶ executive director for food and agriculture of the Biotechnology Industry (BIO), wrote that "BIO supports the overall tone and tenor of the NAS document; we might not be 100% in agreement on some wording and recommendations, but the report is not an indictment of the existing system. … It is a system that is flexible enough to incorporate many of the report's recommendations, which will make it even better."

Risk analysis of transgenic plants must provide technical support for regulatory decision making, and establishment and maintenance of regulatory legitimacy.¹ The NRC report emphasized that appropriate environmental risk analysis and public acceptance of transgenic plants depend on a federal regulatory system and culture that communicates to the public the seriousness with which environmental risks are addressed. APHIS Administrator Acord³ said that the agency "will thoroughly review the report and study its recommendations. We believe that this study will be an important tool in the future regulation of biotechnology."

Sources

1. Committee on Environmental Impacts Associated with Commercialization of Transgenic Plants, National Research Council. 2002. Environmental effects of transgenic plants: The scope and adequacy of regulation. National Academy Press, Washington, DC. http://www.nap.edu/catalog/10258.html.

2. National Academy of Sciences. 2002. Regulation of transgenic plants should be reinforced; Field monitoring for environmental effects is needed. http://www4.nationalacademies.org/news.nsf/isbn/0309082633?OpenDocument.

3. Bobby Acord (Administrator of APHIS, US Department of Agriculture ). 2002. Release No. 0060.02. http://www.usda.gov/news/releases/2002/02/0060.htm.

4. Andrew C. Revkin. 2002. Panel urges US to tighten approval of gene-altered crops. New York Times, February 22, 2002. http://www.nytimes.com/2002/02/22/science/22CROP.html.

5. Randy Fabi. 2002. U.S. biotech crop regulations weak, panel contends. Reuters, http://biz.yahoo.com/rf/020221/n21128806_1.html.

6. Michael J. Phillips, Biotechnology Industry Organization. 2002. BIO statement on NAS report on transgenic plants. http://www.bio.org/newsroom/newsitem.asp?id=2002_0221_03.

Eric M. Hallerman
Department of Fisheries and Wildlife Sciences
Virginia Polytechnic Institute and State University
Blacksburg, VA 24061-0321
ehallerm@vt.edu

At least three other teams of university-based scientists have notified Nature of shortcomings and asked that the paper be withdrawn. Details of these letters are not available due to a press embargo imposed by Nature in mid-December, 2001. However, a leaked internal memo by a Nature referee discussing the Quist and Chapela paper supports the validity of arguments posed by the critics of the paper and further suggests that Nature should consider retracting it (http://www.lifesciencesnetwork.com/news-detail.asp?newsID=676).

In their Nature article, Quist and Chapela used polymerase chain reaction (PCR) and inverse PCR to find the cauliflower mosaic virus 35S promoter (CaMV 35S) sequence in four out of six cobs sampled from two fields in the remote state of Oaxaca in southern Mexico. CaMV 35S is a commonly used sequence in transgenic constructs, and cauliflower mosaic virus is not generally known to infect corn. Consequently, the presence of the CaMV 35S sequence in criollo maize could suggest outcrossing from a transgenic variety.

Methodological criticisms of the Quist and Chapela analysis focus on the mis-interpretation of the PCR and iPCR results. According to Christou's analysis in Transgenic Research, "the authors have not been able to show the presence of intact inserts" of the expected DNA sequences. The authors explain these anomalies by suggesting they are the result of sequence rearrangements during the transformation event or recombination. But Christou adds that rearrangement is an unlikely explanation since the transgenic constructs have been shown to be stable over multiple generations.

Quist and Chapela also report finding sequences "similar to synthetic constructs containing regions of the adh1 gene found currently on the market." However, Christou notes that, in two cases where Quist and Chapela claim to find the adh1 gene sequence, the iPCR results showed the CaMV promoter to be located within the adh1 sequence, not at one end as in the original construct.

Finally, Quist and Chapela claim to have found the nopaline synthase terminator (T-NOS) sequence from Agrobacterium tumefaciens, a commonly used vector for plant transformation, as well as the cry1Ab toxin gene from Bacillus thuringiensis in some samples, but according to several scientists, their paper in Nature does not include enough data about the sequences to substantiate those claims.

Ultimately, many scientists agree that the evidence presented does not support claims of transgene introgression or rearrangements. According to Christou, "It is most likely that the report by Quist and Chapela is a testimony to technical failure and artifacts which are common with PCR and iPCR." He and others believe that the results are the false positives to which PCR and iPCR are prone.

Publication of the Quist and Chapela findings in Nature was widely reported in the popular media around the world. And the authors' allegations that transgene spread represented a grave threat to food security were naturally met with great concern among some environmentalists, politicians, and farmers. Despite reports indicating methodological flaws, noted above, the authors have continued to repeat their original claims.

Using those claims as justification, environmental organizations, including Greenpeace, ETC Group (formerly the Rural Advancement Foundation International), and Friends of the Earth have called for a global moratorium on transgenic crops. In December, the Mexican senate called on President Vicente Fox to ban imports of maize from the United States, and trade analysts suggested that the report could be used by European governments as justification for continuing their moratorium on the approval of new transgenic crop varieties.

Individuals and environmental organizations alarmed by the reports of transgenes in Mexican maize landraces also took issue with the subsequent scientific discourse questioning the validity of these findings. They circulated a petition on the Food First Web site (http://www.foodfirst.org/progs/global/ge/jointstatement2002.html) entitled "Joint Statement on the Mexican GM Maize Scandal" (excerpted below), which, among other things, accuses the scientists who have questioned the Quist and Chapela results of "academic intimidation" and of conducting "a highly unethical mud-slinging campaign."

In response, scientists organized through AgBioWorld Foundation crafted their own "Joint Statement in Support of Scientific Discourse in Mexican GM Maize Scandal" (reproduced below), defending the freedom of scientists to rigorously examine the results and methodology of reported research. The statement (http://www.agbioworld.org/jointstatement.html), with nearly 100 signatures to date, affirms that "relentless double-checking and independent third party evaluations are the cornerstones of the scientific process," and further, "This is in fact how science corrects mistakes and ever more closely approximates truth and understanding."

In the end, most scientists believe it is inevitable that transgenes will eventually be found in maize landraces. Although tests of numerous samples from the International Maize and Wheat Improvement Center (CIMMYT) gene banks showed no evidence of transgenes, preliminary results of other research conducted by the Mexican government's National Commission on Biodiversity already may have found transgenes in both Oaxaca and the neighboring state of Puebla.⁶ Given that level of agreement, the more important question is what will be the effect of transgene spread when it does occur?

Luis Herrera-Estrella, director of the Center for Research and Advanced Studies in Irapuato, Mexico, has noted that "gene flow between commercial and native varieties is a natural process that has been occurring for many decades," so "there is no scientific basis for believing that out-crossing from biotech crops could endanger maize biodiversity."

Mexican farmers reproduce their landraces by carefully selecting the seed they save from year to year. Thus, if a gene producing an undesirable trait is transferred into certain plants, seed from those crops will not be planted the following year and will be eliminated from the gene pool. Mexican corn biologists Juan Pablo Martinez-Soriano and Diana Leal-Klevezas have written that, "Any transgene transferred inadvertently to native maizes can be removed from the progeny by selecting against the incorporated trait." This practice has worked very well for millennia and explains why Mexican farmers can plant many different landraces next to one another without worrying about cross-pollination.

Journalist Ronald Baileysums up the issue succinctly: "Two questions arise from the Nature study: Is it true? And does it matter?" In this case, the answer to both questions appears likely to be "no."

Sources

1. Quist D and Chapela IH. 2001. Transgenic DNA introgressed into traditional maize landraces in Oaxaca, Mexico. Nature 414: 541–543.

2. Yang S. 2001. Transgenic DNA discovered in native Mexican corn, according to a new study by UC Berkeley researchers. University of California at Berkeley press release (November 29), available at: <http://www.berkeley.edu/news/media/releases/2001/11/29_corn.html>.

3. See, for example, Chapman J. 2001. GM crops `pollute 60 miles away'. The Daily Mail (November 29): 42.

4. Manning A. 2001. Gene-altered DNA may be `polluting' corn. USA Today (November 29), available at: <http://www.usatoday.com/news/healthscience/science/biology/2001 -11-28-biofood-mexico.htm>.

5. Christou P. 2002. No Credible Evidence is Presented to Support Claims that Transgenic DNA was Introgressed into Traditional Maize Landraces in Oaxaca, Mexico. Transgenic Research 11: iii-v.

6. Hodgson J. 2002. Maize uncertainties create political fallout. Nature Biotechnology 20: 106–107.

7. Zarembo A. 2002. The Tale of the Mystery Corn in Mexico's Hills. Newsweek International (January 28): 50.

8. Hodgson J. 2002. Doubts linger over Mexican corn analysis. Nature Biotechnology 20: 3–4.

9. In AgBioWorld Foundation. 2001. Scientists Say Mexican Biodiversity Is Safe: Concerns About Cross-Pollination Unfounded. AgBioWorld Foundation Press Release (December 19).

10. Loutte D and Smale M. 2000. Farmer's seed selection practices and traditional maize varieties in Cuzalapa, Mexico. Euphytica 113: 25–41.

11. Martinez-Soriano JPR and Leal-Klevezas DS. 2000. Transgenic Maize in Mexico: No Need for Concern. Science 287: 1399.

12. Bailey R. 2002. Environmentalist Biofraud? `A new report challenges research published in the respected journal, Nature'. Reason (February 12). Available at: <http://reason.com/rb/rb021202.shtml>.

Gregory Conko Competitive Enterprise Institute Washington DC conko@cei.org

C. S. Prakash Tuskegee University Tuskegee, AL prakash@tusk.edu

Note: Prakash and Conko are co-founders of the AgBioWorld Foundation (http://www.agbioworld.org)

JOINT STATEMENT ON THE MEXICAN GM MAIZE SCANDAL

We call upon CIMMYT to:
Publicly acknowledge that GM maize contamination has taken place in Mesoamerica; Confirm that under present circumstances the operational assumption has to be that GM maize contamination in gene banks is inevitable;

We call upon FAO to:
•Ensure that the Code of Conduct on Biotechnology which is currently under development, incorporate mechanisms to control the diffusion of GM materials to vulnerable regions and to guarantee that the burden of ecosystem restoration and farmer and national compensation rests with those who pollute.

We call upon CGIAR and FAO together to:
•Review the current FAO-CGIAR Trust Agreement to ensure that the integrity of germplasm held in Trust is protected and that there are no intellectual property claims pertaining to any of the germplasm;
•Recommend steps to safeguard local farmers' varieties and gene banks.
•Propose an immediate moratorium on the shipment of GM seed or grain in countries or regions that form part of the Centre of Origin or Centre of Genetic Diversity for the species.

We call upon Academia and the Private Industry to:
•Renounce immediately the use of intimidatory tactics to silence potentially `dissident' scientists. We call upon the scientific community to publicly support the academic freedom of scientists whose studies conflict with the interests of industry and to censor those academics and institutions that slander the competence or integrity of those who publish peer-reviewed studies.

---

Recently, several activist organizations and individuals signed a "Joint Statement" charging impropriety and criticizing vigorous scientific debate surrounding controversial GMO research published in Nature. The research supposedly demonstrated that Mexican landrace maize varieties had been "contaminated" with genetic material from maize varieties improved through biotechnology, presumably through cross pollination (activist statement available at http://www.foodfirst.org).

It is important to recognize that the kind of gene flow alleged in the Nature paper is both inevitable and welcome. It is inevitable because of the nature of maize, and it is welcome as demonstrated by the standard practices landrace custodians have used to improve their varieties for thousands of years—increasing variation by planting seeds of new varieties adjacent to old ones, and then selecting the desired offspring while discarding the rest.

However, several scientists have now challenged the methodology and the results reported in the Nature paper in formal letters to Nature. The editorial board of the journal Transgenic Research found it surprising "that a manuscript with so many fundamental flaws was published in a scientific journal."

These challenges are based on the fact that the key research method employed is highly prone to false positives, and the Nature paper failed to use standard techniques to ensure accuracy and confirm results. The "joint statement" signed by the activists strongly condemns these challenges from fellow scientists as nothing more than "academic intimidation" and "a highly unethical mud-slinging campaign."

It must be stated clearly and unequivocally: scientists have a fundamental ethical obligation to rigorously examine the results and methodology of reported research. This is in fact how science corrects mistakes and ever more closely approximates truth and understanding. Far from being "mudslinging" or "intimidation," all scientists worthy of the name understand that relentless double-checking and independent third party evaluations are the cornerstones of the scientific process.

Such relentless criticism and re-examination is perhaps most important when it leads in directions that may conflict with a point of view driven by politics or activism, rather than science.

We the undersigned scientists declare our support for appropriate and necessary scientific discourse and debate, especially in areas marked by widespread misunderstanding and misrepresentation, such as agricultural biotechnology.

(Signature list available at http://www.agbioworld.org/jointstatement.html.)

The results are discussed at length in a feature story that appears in the February issue of Agricultural Research magazine. ARS, the US Department of Agriculture's primary scientific research agency, also has a Web site about the Bt corn-monarch butterfly issue at: http://www.ars.usda.gov/is/br/btcorn

Bt corn is corn to which genes from the bacterium Bacillus thuringiensis have been added so the plant naturally produces proteins that protect it from insect pests such as the European corn borer.

The research found that Bt corn pollen levels usually had to be more than 1,000 grains per square centimeter to have any negative impact on monarch caterpillars, let alone mortality. Scientists have concluded that monarch caterpillars in the environment are exposed to levels that come close to that magnitude less than one percent of the time.

ARS entomologist Richard Hellmich is already planning the next round of investigations. He hopes to extend the consortium's work this summer with new collaborative studies, especially field studies, to look at whether there are any effects on monarch caterpillars from long-term or chronic exposure to Bt corn pollen. While the data already accumulated show Bt corn pollen does not pose a threat to monarch populations, these new studies should indicate if any minor effects are possible and the nature of those effects if they occur. ARS entomologist Leslie C. Lewis is planning to extend the work to look at whether Bt corn has any impact on non-target ground insects such as beetles. Hellmich and Lewis are both with the ARS Corn Insects and Crop Genetics Laboratory in Ames, Iowa.

You can read more about this in the February issue of Agricultural Research magazine, available on the World Wide Web at: http://www.ars.usda.gov/is/AR/archive/feb02/corn0202.htm

Kim Kaplan
ARS News Service
Agricultural Research Service, USDA
Kaplan@ars.usda.gov

EDIBLE FOOT AND MOUTH VACCINATION FROM TRANSGENIC PLANTS

Plants are aggressively investigated as potential hosts for vaccine production, in part to avert the chance of transferring undetectable animal viruses and prions into the product. Other benefits include the reduced potential for adverse reactions, economy, stability, low storage costs, and the potential to be administered either parenterally or as edible vaccines. Though vaccines for human viruses were the focus of early endeavors, now investigators are increasingly using plants for the production of animal vaccines.

Manuel Borca's research team, at the Instituto de Virologia in Buenos Aires, Argentina, has been investigating the use of transgenic plants as vectors of edible vaccine against hoof and mouth disease (HMD) in mice, and eventually plans to apply the results of this research to cattle. Initial studies demonstrated success in expressing the immunogenic agent for HMD in alfalfa and potato, however the level of expressed protein was too low to be effective when administered to mice as an edible product. One strategy for increasing the concentration of the immunogenic antigen is to identify those transgenic plants in which it is expressed at an exceptionally high level. To that end, Borca and colleagues report in the journal Vaccine that they have successfully fused an easily detectable reporter gene, gus A, to the antigenic portion of the foot and mouth disease virus (FMDV), VP1.¹ Consequently, they were able to readily screen a large number of plants for high level of VP expression and demonstrate immunity against virulent FMDV in mice injected with extracts of the forage crop.

In a host of studies conducted between 1993 and 1999, various investigators report they have identified and isolated the viral proteins that elicit an immune response to the FMD virus. Drawing upon these studies as well as their own work, Borca and colleagues chose the viral structural protein VP1 as the antigen most likely to be effective as an oral vaccine² because of its ability to induce a strong and specific immune response against FMDV in host animals (refer also to Nunez JI et al. 2001. Veterinary Research 32: 31–45). They also determined the parameters for the dose of enterically administered vaccine needed to provide effective protection against FMD.³ The VP1 capsid protein has a conserved arginine-glycine-aspartate sequence on a variable loop used for host binding. Antibodies against bacterial and viral attachment proteins are well suited for vaccine production, assuming the domain coding for the protein is not highly variable. In general, a drawback to using attachment proteins is that they often have a high degree of host specificity, which limits the scope of the vaccine. Fortunately, the VP1 protein appears to have a wide host spectrum, making it acceptable for use in the entire range of mammals infected by or carrying FMDV.

In 1998, Borca's team expressed VP1 in Arabidopsis thaliana using a synthetic amino acid sequence similar to the FMDV VP1 gene. After determining successful expression of the sequence with Western blotting, the team intraperitoneally immunized mice with leaf extracts. The mice showed a strong immune response as evident by antibody titer and protection when inoculated with FMDV.⁴ A similar study was conducted in 1999 in which Borca and colleagues expressed VP1 in Nicotiana benthamiana and again successfully protected mice with intraperitoneal injections of the transgenic foliar extracts (Wigdorovitz et al. 1999. Virology 264(1): 85–91). More recently, they induced immunity to FMD from VP1 expressed in potatoes, Solanum tuberosum (Carrillo et al. 2001. Viral Immunology 14(1): 49–58). In spite of these successes, it remained difficult to obtain antigen expression at levels sufficient to produce a highly effective edible vaccine.

Borca's most recently reported investigation involves strategies for intensifying antigen expression in plants without sacrificing the exactitude of the vaccine. Simultaneously, he was hoping to develop a technique that was simple and inexpensive. By fusing amino acid residues 135–160 of the structural protein VP1, known for its high degree of antigenicity, to ß-glucuronidase (ßGUS), Borca and colleagues claim they discovered an uncomplicated technique for identifying transgenic plants expressing large amounts of vaccine. In this arrangement, plants expressing the highest level of VP135–160 were easily selected based on expression of ßGUS enzymatic activity.¹

As in previous studies, the vaccine elicited FMD antibody production in mice immunized intraperitoneally with extracts from the highest-expressing plants, which contained a VP–ßGUS concentration of between 0.5 and 1 mg/g of total soluble protein. The mice not only displayed significant antibody production, but exhibited 100% protection against foot and mouth disease, made evident when the mice were challenged with infectious FMDV. These results also demonstrate that synthetic vaccine peptides appear to work as well as the natural peptides when expressed in transgenic plants, which simplifies vaccine preparation for a variety of exotic parasites and pathogens. Indeed, Borca et al. claim that this trial is the first study showing complete protection against disease using a peptide-based vaccine produced in transgenic plants.

Similar plant vaccine trials were performed in 1998 by Francisco Parra's team (Instituto Universitario de Biotecnología de Asturias, Universidad de Oviedo, Spain).⁵ They expressed VP60, a structural protein effective in eliciting immunity to rabbit hemorrhagic disease virus (RHDV), in transgenic potatoes. To do this, they inserted the VP60 coding region of RHDV into a pRok2 plasmid controlled by a 35S promoter. Rabbit trials indicated that the vaccine was most effective in inducing immunity to RHDV when plant extracts were injected into the subjects. They concluded that plants expressing higher titers of vaccine would have to be produced for successful oral delivery.

More recently, a research team led by John Howard at ProdiGene (College Station, Texas) report in 2001 the first known occurrence of a conventional food animal acquiring protection from a major disease, swine transmissible gastroenteritis (TGE), through ingesting an edible vaccine engineered into feed corn.⁶ They expressed the structural surface protein subunit (S) of the highly contagious TGE virus in corn by fusing the maize codon for the S protein to a barley alpha-amylase signal sequence known to result in high levels of protein accumulation in the cell wall. Half the piglets fed the transgenic corn exhibited TGE clinical symptoms, compared to 100% of the piglets fed wild-type corn and 78% of those receiving a commercial modified live vaccine. However, those receiving the commercial vaccine recovered more quickly than those fed the transgenic corn. In a concurrent study, the researchers were also able to demonstrate immunogenicity in mice fed transgenic corn expressing the binding subunit of the E. coli heat-labile (LT) toxin, which is responsible for 800 000 deaths each year in children under age five in developing countries, as well as "travelers diarrhea" in 20% of visitors to these countries. In this case, they constructed a plasmid consisting of a synthetic version of the gene for the LT-B subunit, again optimized by incorporation of the alpha-amylase signal sequence. Surprisingly, the mice exhibited a much stronger Ig response to the LT-B expressed in corn than to purified LT-B, which demonstrates the effectiveness of corn for oral delivery.

Studies such as those by Borca, Howard, and others further the likelihood of inexpensively producing edible plant vaccines for human as well as animal consumption and may eventually lead to an improved ability to prevent disease epidemics in agricultural and domestic animals, as well as control outbreaks such as rabies in wildlife and reservoir species. The threat of agricultural bioterrorism may also necessitate large-scale and efficient vaccination regimens capable of protecting animals from biological agents, for which transgenic vaccine-containing plants would be ideally suited.

Charles Arntzen, of Arizona State University, who did some of the preliminary analyses of edible vaccines in plants (see: http://www.nal.usda.gov/pgdic/Probe/v5n1/lead.html), asserts that animals are a likely target for plant-produced vaccine applications. However, Arntzen and others caution that repeated low-dose oral vaccination may lead to "oral tolerance," which renders the immune system less sensitive to the disease agent and suppressing antibody production.

Sources

1. Dus Santos MJ, Wigdorovitz A, Trono K, Ríos RD, Franzone PM, Gil F, Moreno J, Carrillo C, Escribano JM and Borca MV. 2002. A novel methodology to develop a foot and mouth disease virus (FMDV) peptide-based vaccine in transgenic plants. Vaccine 20 (7–8): 1141–1147.

2. Wigdorovitz A, Carrillo C, Dus Santos MJ, Trono K, Peralta A, Gómez MC, Ríos RD, Franzone PM, Sadir AM, Escribano JM and Borca MV. 1999. Induction of a protective antibody response to foot and mouth disease virus in mice following oral or parenteral immunization with alfalfa transgenic plants expressing the viral structural protein VP1. Virology 255: 347–353.

3. Sadir AM, Zamorano PI, Romera A, Wigdorovitz A, Smitsaart E, Marangunich L, Schiappacassi C and Borca MV. 1999. Improvement of the immune response to foot and mouth disease virus vaccine in calves by using Avridine as adjuvant. Veterinary Immunology and Immunopathology 69: 11–22.

4. Carrillo C, Wigdorovitz A, Oliveros JC, Zamorano PI, Sadir AM, Gomez N, Salinas J, Escribano JM and Borca M. 1998. Protective immune response to foot-and-mouth disease virus with VP1 expressed in transgenic plants. Journal of Virology 72(2): 1688–1690.

5. Castañón S, Marin MS, Martín-Alonso JM, Boga JA, Casais R, Humara JM, Ordas RH and Parra F. 1999. Immunization with potato plants expressing VP60 proteins protects against rabbit hemorrhagic disease virus. Journal of Virology 73(5): 4452–4455.

6. Streatfield SJ, Jilka JM, Hood EE, Turner DD, Bailey MR, Mayor JM, Woodard SL, Beifuss KK, Horn ME, Delaney DE, Tizard IR, and Howard JA. 2001. Plant-based vaccines: unique advantages. Vaccine 19: 2742–2748.

Brian R. Shmaefsky
Department of Biology and Environmental Sciences
Kingwood College
brian.shmaefsky@nhmccd.edu

MAMMALIAN CELLS EXPRESS SPIDER SILK PROTEINS

No synthetic fiber matches the unique combination of strength and flexibility of spider dragline silk, which is used as the safety line and the framework for the spider's web. Spider dragline silk is a flexible, lightweight fiber that is stronger than steel on a weight basis and stretches better than nylon. Because of these properties, scientists have attempted to artificially manufacture spider silk proteins in bacteria and yeast systems but have met with only limited success.

In the January 18, 2002, issue of Science, researchers at Nexia Biotechnologies (Quebec) and the US Army Soldier Biological Chemical Command (Natick, MA) report they have teamed up to produce soluble spider silkline protein in cultured mammalian cells. Dragline silk genes from two species of spiders were stably introduced into two cell lines: bovine mammary epithelial (MAC-T) cells and baby hamster kidney (BHK) cells. In both cases, these cell lines were chosen because of their ability to express and secrete foreign proteins.

MAC-T and BHK cells both secreted into the media silk line proteins of the correct molecular size that were recognized by antibodies to the silk proteins. In large-scale cell culture experiments, spider silk proteins from BHK cells were secreted at 25-50 mg/liter corresponding to 20 µg per million cells per day. More than 12 g of material was purified from the cultured media by precipitation with ammonium sulfate and redissolving in phosphate buffered saline. This is in marked contrast to recombinant silk proteins previously produced in bacteria or yeast, which were soluble only in strong denaturing solvents.

This soluble spider silk protein solution was subsequently used to spin insoluble fibers, by extruding the solution into a methanol bath through a fine piece of tubing that acted as a spinneret. Microscopic analysis of the fibers revealed fibers with a cylindrical, smooth surface and a solid inner core. Analysis of the mechanical properties of these fibers showed that their toughness compared favorably with that of native silk although their tenacity values were lower than native silk. This latter result may be due to the fact that the manufactured fibers were composed of only one spider silk protein, whereas native spider silk consists of two.

The unique mechanical properties of spider dragline silk have been coveted for a number of biomedical and industrial applications. These fibers could be used for wound closure systems, such as patches, glue, and ultrafine sutures for microsurgery. Spider silk has also been proposed for soft body armor applications, because it is thought to be tougher and lighter than the currently widely used Kevlar.

Because of their experience in producing transgenic animals as bioreactors, Nexia is strategically positioned to bioengineer the mammary gland of transgenic goats for scale-up production of spider silk line protein. The bovine Mac-T cells were specifically used in these preliminary studies because they are secretary epithelial cells similar to the types of cells that express silk proteins in the spider gland and because they might provide some indication of the ability of mammary epithelial cells to secrete silk proteins in the milk of transgenic animals. Perhaps in the not so distant future we will be reading about the adventures of a real superhero, Spidergoat.

Source

Lazais et al. 2002. Spider silk fibers spun from soluble recombinant silk produced in mammalian cells. Science 295: 472–476.

Eric A. Wong
Department of Animal and Poultry Sciences
Virginia Tech
ewong@vt.edu

INVENTORS HASH OUT PRIORITY FOR CLONING TECHNOLOGY AND OTHER BIOTECH LAW NEWS

"All animals are equal. But some animals are more equal than others." George Orwell, Animal Farm (1945)

A dispute over inventorship has rammed a herd of cloned animals into the limelight: Dolly the sheep (Roslin Institute), Gene the cow (Infigen, Inc.), and bovine buddies, George and Charlie (Advanced Cell Technology). On January 30, the US Patent and Trademark Office declared an interference between a patent and a patent application claiming the use of nuclear transfer technology to produce non-human mammals. The patent (US patent No. 5,945,577; "Cloning Using Donor Nuclei from Proliferating Somatic Cells") is assigned to the University of Massachusetts and exclusively licensed to Advanced Cell Technology (Worcester, MA), and the patent application (US Serial No. 09/650,194) is assigned to the Roslin Institute (Scotland UK) et al., and licensed to Geron Corporation (Menlo Park, CA). The interference began when the PTO granted Geron's request for the proceeding. Infigen, Inc. (DeForest, WI) had also requested an interference, and reportedly has become a third party in this proceeding to determine who was the first to invent the method for cloning mammals from non-embryonic cells.

Interference practice is unique to the United States, which has a patent system that grants patents to the first party to invent the claimed subject matter. The interference proceeding is the mechanism by which the PTO determines who invented the claimed technology first. In an interference, the first party to file the patent application is designated as the "senior party," and all other parties are designated as "junior" parties. According to interference rules, there is a presumption that parties made their inventions in the order of the filing dates of their patent applications. Consequently, a junior party has an initial burden of proving a date of invention before the senior party. The PTO may take years to determine which among the three sets of cloning technology inventors is the original inventor.

This is not an academic exercise. The disputed technology has potential uses in human therapy and in the production of protein pharmaceuticals. In addition, the market for cloned animals is expanding. The U.S. Food and Drug Administration may allow the sale of clone-derived meat and dairy products this year. On the domestic front, the recent announcement by Texas A&M researchers of the first cloned cat has bolstered the promise of companies like Genetics Savings & Clone (College Station, TX), which aims to clone cherished pets.

The Other Biotech Law News . . .
Last December, the Standing Committee on Health of the Canadian House of Commons released a report ("Assisted Human Reproduction: Building Families"), which included a recommended ban on gene patenting (copy available at the website of Canada's Parliament: http://www.parl.gc.ca/InfoComDoc/37/1/HEAL/Studies/Reports/healrp01/06-toc-e.htm). Taking a more balanced approach, the Ministry of Health and Long-Term Care (Ontario, Canada) recently published its draft report entitled "Genetics, Testing & Gene Patenting: Charting New Territory in Healthcare." The Ministry does not call for a ban on gene patenting, but instead recommends a review of the Canadian Patent Act, and possible infringement protection for healthcare providers and a restriction on certain broad patents. A copy of the report is available from the Ministry's website (http://www.gov.on.ca/health/english/pub/ministry/geneticsrep02/genetics.html).

Patenting is receiving the attention of the US government, as well. In the near future, a bill will reportedly be introduced in the House of Representatives, which would instruct the Director of the Office of Science and Technology Policy to perform a study on the impact of federal policies on the innovation process for genomic technologies. One of the concerns, here, is that current federal policies may inhibit basic research. Coincidentally, John Walsh (University of Illinois, Chicago) recently described the results of his survey on biotech patenting at a NAS-sponsored conference on the effects of patenting on research. One finding was that university researchers feel free to use patented technologies and that companies generally refrain from asserting infringement actions against the researchers. The IP@The National Academies website (http://ip.nationalacademies.org/) offers a transcript of the proceedings.

The Federal Trade Commission and the Antitrust Division of the Department of Justice have been holding hearings on the implications of competition and patent law for innovation. The sessions, which began in February and may continue through June, are open to the general public on a first-come, first-served basis. Issues under consideration include: the role of intellectual property in fostering innovation, licensing of intellectual property, patent examination procedures, and the settlement of patent disputes. Information about the hearings can be found on the FTC's website (http://www.ftc.gov/opp/intellect/index.htm).

Selected References on the Cloning Claim Clash:

Correia-Moreno M. 2002. Which came first, the cow or the sheep–the U.S. Patent Office to decide. (February 1). Available: http://www.prnewswire.com.

Mishra R. 2002. Patent dispute getting woolly. Boston Globe A1 (February 19).

Zobkiw L. 2002. U.S. Patent Office grants Geron's request for an interference with U.S. Patent No. 5,945,577. (January 31). Available: http://www.geron.com.

Phillip B. C. Jones, PhD., J.D.
Seattle, Washington
phillipjones5939@msn.com

More meetings can be found at http://www.isb.vt.edu

First International Congress on Plant Metabolomics
April 7 - 11, 2002
Wageningen, The Netherlands

This is the first international congress dedicated to plant metabolomics and will foster discussion on plants as the source of pharmaceuticals, health-promoting compounds, flavour & fragrance compounds, protectants, biocides, fine chemicals, toxins, etc. Session topics are:

• Metabolic Profiling
• Genomics, Proteomics, Metabolomics
• HTP-screening
• Bio-informatics
• Food and Health
• Harvesting Metabolomics

Contact: Emmy van Balen
Fax: +31 317 41 80 94
Email: E.vanBalen@plant.wag-ur.nl
http://www.metabolomics.nl/

Biosafety 3 – "Advanced Issues on Biosafety: Risk Monitoring and Public Perception of Biotechnology"
May 13–17, 2002
Maracay, Venezuela

General aspects of biosafety and risk assessment, risk monitoring of GMOs, and public perception of biotechnology will be the issues addressed by international experts in this workshop.

Contact:

Dr. Efrain G. Salazar Yamarte
Tel: +58-243-2471066
Fax: +58-243-2471066
Email: efra63@icnet.com.ve
http://www.icgeb.trieste.it/~bsafesrv/bsfn0112.htm

ISB News Report
207 Engel Hall
Virginia Tech
Blacksburg, VA 24061

The material in this News Report is compiled by NBIAP's Information Systems for Biotechnology, a joint project of USDA/CSREES and the Virginia Polytechnic Institute and State University. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture, or Virginia Tech. The News Report may be freely photocopied or otherwise distributed without charge.

ISB welcomes your comments and encourages article submissions. If you have a suitable article relevant to our coverage of the agricultural and environmental applications of genetic engineering, please e-mail it to the Editor for consideration.

Ruth Irwin, Editor (rirwin@vt.edu)

To have the News Report automatically e-mailed to you, send an e-mail message to news@nbiap.biochem.vt.edu and type subscribe newsreport [your name] in the message section. Do not include a signature file or additional text. To unsubscribe, send e-mail to news@nbiap.biochem.vt.edu and type unsubscribe newsreport [your name] in the message section, or e-mail isb@vt.edu with your request.
Connect to http://www.isb.vt.edu for internet access to ISB News Reports, textfiles, and databases.

Information Systems for Biotechnology, 207 Engel Hall, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, tel: 540-231-3747, fax: 540-231-4434, e-mail: isb@vt.edu