By Katharina Schoebi, Checkbiotech

Recently many politicians in the US and the EU have received Anthrax-laced envelopes. The threat of biological warfare and terrorism is real; Anthrax is just one of many potential bio attack agents, but it is a serious one. Since the only vaccine existing so far against Anthrax has some disadvantages, researchers are now developing a new one.

Anthrax is endemic in the US and is caused by the bacterium Bacillus anthracis, which infects humans in one of three ways: through the skin (cutaneous anthrax), through consumption of contaminated meat products (gastrointenstinal anthrax) and through the nasopharynx (inhalational anthrax). However, once in the human body the bacteria produce toxins that lead to problematic symptoms and possible death.

The Centers for Disease Control (CDC) lists anthrax as a category A disease agent with easy dissemination or transmission, high mortality, social disruption and a special preparation needed. Nowadays, biological warfare and terrorism is a real threat. The CDC estimates that the cost of an anthrax attack would be US$ 26.2 billion per 100,000 persons exposed.

The only vaccine licensed so far for human use in the US is Biothrax, which unfortunately has some disadvantages: it has to be injected several times, causes local reactions at the site of the subcutaneous injection and could also lead to side-effects in some individuals. Therefore, researchers are looking for ways, by which they could produce a new vaccine on a large scale and at reasonable cost, which also would be safer.

An economical and convenient way to obtain pharmaceutical products, without human or animal pathogen contamination, is to let plants produce them. In plants, not only the nucleus contains DNA but also the chloroplasts. Chloroplasts are the organelles the plant needs for the photosynthesis. Since they are very advantageous for genetic engineering, they are often used in plant science experiments. If you insert a gene of interest in the DNA of the chloroplasts, you will have two copies of that gene when the chloroplasts divide (when the plant is growing). Bearing in mind that there are up to 10,000 copies of chloroplast genomes per cell, you will obtain quite a large amount of the gene of interest by cultivating plants—much more than you would with the genome of a plant.

One disadvantage of producing pharmaceutical proteins in plants is the possibility of plant-based pharmaceuticals mixing with food supplies. To prevent this, scientists have turned to to producing the pharmaceutical products in non-food, or feed crops such as tobacco (Nicotiana tabacum).

Tobacco is often used plant in biological experiments, because it has many advantages: It is a self-pollinating crop with up to one million seeds per plant, it can produce a large amount of biomass (more than 40 t fresh leaf weight/acre), it has no known wild or cultivated relatives in North America and it is easy to enhance through genetic engineering. By harvesting tobacco leaves before the onset of flowering, transgene flow via pollen or seed is eliminated and the contamination of food crops is prevented. In addition, there exists a large-scale processing infrastructure.

Two researcher teams headed by Henry Daniell from the Department of Molecular Biology and Microbiology at the University of Central Florida in Orlando, USA, and Stephen H. Leppla from the Microbial Pathogenesis Section at the National Institute of Allergy and Infectious Diseases, produced the Bacillus anthracis protective antigen (PA) in chloroplasts of tobacco.

In their experimental studies, they obtained a total of 172 mg of full functional PA from each plant. Extrapolating that out, that would mean that about 8,000 tobacco plants could be grown in an area of one acre. Each year a total amount of 4.12 kg of PA could be expected, out of which 50% would be lost during purification. Taking into consideration that about 5 ìg PA are used per dose of the vaccine, researchers could produce about 400 million doses of vaccine per acre of tobacco. That number might jump to well over 3 billion vaccines, when a commercial tobacco variety is used, in the place of the laboratory variety that Dr. Daniell and Dr. Leppla used.

Having produced the new antigen, the two research teams then had to test its ability to be used as an effective vaccine. This was determined by its ability to cause lysis of cultured mouse macrophages, which is a first step that immunologists use to determine whether or not a vaccine will work or not. Thus the two research teams headed by Dr. Daniell and Dr. Leppla showed that the tobacco plants can be used to produced fully functional antigen that can then be used as a human vaccine.

“Several groups have estimated plant derived vaccines to be 50-100 fold cheaper than those currently produced via fermentation”, said Dr. Daniell. So the new vaccine against bacterium anthracis would be very advantageous.

Equally important is the longevity of a vaccine. It is often very beneficial to have a vaccine that can be prepared in advance, and stored until it is needed. Dr. Daniell and Dr. Leppla were also able to show that there is no degradation of the PA vaccine upon storage and transportation, which further points to their PA from tobacco plants as a very favorable Anthrax vaccine.

However, before using the new Anthrax vaccine, the laboratories of Dr. Daniell and Leppla will have to carry out some future testing before the vaccine will ready to used for humans.

Chlorogen Inc. had already obtained USDA permits to grow tobacco plants with Bacillus anthracis protective antigen field trials, as Dr. Daniell said. It is now under large scale production. But to bring the project closer to commercial use, clinical trials need to be carried out in order to identify, which is the most expensive part of the experiment. “It would cost about $50 millions. Therefore, Chlorogen Inc. is now seeking a partner for commercial production.”

To produce edible plant vaccines, they have to be produced in large quantities in edible parts. Recently, Chlorogen Inc. achieved this breakthrough and several vaccine antigens are now produced in carrots in Dr. Daniell’s laboratory.

The developments in Dr. Daniell’s laboratoy showed that through the cultivation of tobacco plants on a few acres, the worst case senerio from an anthrax attack can be prevented. And as Dr. Daniell pointed out, most certainly it would be possible to eliminate a Bacillus anthracis threat due to his research.

Katharina Schoebi is a biologist and a Science Writer for Checkbiotech. Contact her at katharina.schoebi@bluemail.ch.