10. Tumor- Busting viruses.

A new technique called virotherapy harnesses viruses, those banes of humankind, to stop another scourge-cancer.

Some scientists are now genetically engineering a range of viruses that act as search-and-destroy missiles: selectively infecting and killing cancer cells while leaving healthy ones alone. This new strategy, called virotherapy, has shown promise in animal tests, and clinical trials involving human patients are now under way. Researchers are evaluating virotherapy along and as a novel means for administering traditional chemotherapies solely to tumor cells. They are also developing methods to label viruses with radioactive or fluorescent tags in order to track the movement of the viral agents in patients.

One of the first inklings that viruses could be useful in combating cancer came in 1912, when an Italian gynecologist observed the regression of cervical cancer in a woman who was inoculated with a rabies vaccine made from a live, crippled from of the rabies viruses. Physicians first injected viruses into cancer patients intentionally in the late 1940, but only a handful appeared to benefit.

The modern concept of virotherapy began in the late 1990, when researchers led by Frank McCormik of ONYX Pharmaceuticals in Richmond, California, and Daniel R. Henderson of Calydon in Sunnyvale, California, independently published reports showing they could target virotherapy to human cancer cells grafted into mice; thereby eliminating the human tumors.

Both used adenovirus, a cause of common cold that has been intensively explored for virotherapy. Adenovirus is appealing in part because researchers understand its biology very well after years of truing to cure colds of using the virus in molecular biology and gene therapy research. It consists of a 20-sided protein case, or capsid, filled with DNA and equipped with 12 protein "arm". These protrusions have evolved over millennia to lintch onto a cellular receptor whose normal function is to help cells adhere to one another.

Adenoviruses are distinct from the types of the viruses usually used in gene therapy to treat inherited disorders. Gene therapy traditionally employs retroviruses to splice a functioning copy of a gene permanently into the body of the patient in, however, adenoviruses do not integrate their DNA into the genes of cells they infect; the genes they terry into a cells usually work only for a while and then break down. Scientists have investigated adenoviruses extensively in gene therapy approaches to treat cancer, in which the viruses are armed with genes that, for example, make cancer cells more susceptible than normal ones to chemotherapy. In general, test involving adenoviruses have been safe, but regrettably a volunteer died in 1999 after receiving an infusion of adenoviruses as a part of a clinical trial to test a potential gene therapy for a genetic liver disorder.

11. Vaccine Cuisine

In a field on the west coast of Africa, banana trees stand some 25 feet tall. The trees huge frond like leaves shadow clusters of ripening fruit, peels green in the midday sun. Each tree supports up to 150 bananas, enough to feed a crowd of people.

In coming years, tree like these may provide more than sweet nutrition. Bananas could become the world’s first edible vaccine. “It may sound unusual,” says Charles Arntzen, a plant biologist and president of Cornell University’s Boyse Thompson Institute for Plant Research. “But you have to realize that many people in the developing world don’t have access to agriculture. So plants could be a way to deliver vaccines.”

According to the World Health Organization (WHO), more than 2 million children – most in the developing world – die each year from diseases that can be prevented with vaccines. Bacterial diarrheas, which sicken adults but can dehydrate and kill children, are a major problem. Researchers at Boyce Thompson hope to develop oral plant vaccines to prevent deadly diarrhea caused by Escherichia coli and Vibrio cholera bacteria.

Like injected vaccines, the edible variety would not cause disease, but would train the body’s immune system to recognize and attack a disease bacteria or virus. Unlike injected vaccines, however, the edible ones would be inexpensive and easy to distribute.

“Current recombinant vaccines are expensive because they need fermentation and protein purification,” explains Hugh Mason, a molecular biologist at Boyce Thompson. “If you can instead produce the immunogenic protein in an edible plant tissue, you can lower the cost. I can envision growing large plots of plants in a way that makes [vaccine] production very cheap.”

Scientists at Boyse Thompson hope to use a straightforward bacterial vector approach to engineer life-saving bananas. First, they will splice the gene for an immune system-stimulating disease protein into Agrobacterium. When exposed to banana cells, the Agrobacterium will shuttle the gene inside. Researchers will then grow the cells into mature plants bearing fruit that, when eaten, confer immunity to the particular disease.

These researchers have already used this technique to engineer potatoes with a modified E. coli protein from a bacterial strain that normally causes severe diarrhea. Eating the raw potatoes, mice developed antibodies to the E. coli toxin. Theoretically, humans who eat the potatoes should develop similar immunity. “We’ve demonstrated feasibility,” Arntzen says. “Now, we want to demonstrate efficacy.” Arntzen and collaborators at a national vaccine testing center hope begin human clinical trials with the E. coli-carrying potatoes this year.

“It’s very exciting work,” says Joseph St. Geme, a pediatrician at Washington University School of Medicine in St. Louis, Missouri. “There is increasing evidence that the oral immunization route is going to be effective. And it’s as likely to be effective with a banana as with any other oral method of introducing an antigen.”

At Scripps Research Institute in La Jolla, California, plant biologist Mich Hein is also studying oral vaccine delivery. Hein is growing alfalfa spiked with a modified cholera toxin, which should act as a vaccine when fed to mice. He hopes to study the highly immunogenic cholera toxin as a prelude to other diseases, particularly those that affect cattle.

“If we can find out what makes the modified cholera toxin an active oral vaccine in mice, we might be able to translate that into other diseases and other animals,” Hein explains. “For example, it could be cheaper for the agriculture industry to deliver vaccines this way. And once we have addressed the safety and efficacy issues in nonhuman populations, we could move on to people.”

John Clements, a microbiologist at Tulane University who collaborates with Arntzen, agrees. “The good thing about cattle or sheep is that you can control their daily diet,” Clements adds. “You can clone an antigen into their alfalfa or hay feedstock, and the animals will consume large, consistent quantities. It’s very practical.”

Ultimately, the researchers envision officials in the developing world distributing fresh vaccine-carrying fruit to villagers. Medicinal bananas might sport a different colored peel – thanks to an engineered pigmentation gene – that distinguished it from normal fruit. “With simple distribution,” Arntzen says, “we might be able to do a lot of good.”

The Perfect Vaccine. In 1992, WHO and other international organizations launched the Children’s Vaccine Initiative, a cooperative effort to promote vaccine research for the developing world. That year, during a trip to Thailand, Arntzen was standing near a floating market when he noticed a mother running her finger along the top of a banana, lifting a taste of the fruit to her infant’s lips. The scene stayed with him. “It just seemed so obvious to me,” says Arntzen, then at Texas A&M University. “Bananas would be the perfect food for an oral vaccine.” The fruit is cheap, abundant, and often fed to children, he notes. “And most importantly, it is eaten uncooked, so the vaccine would not be destroyed by heating.”

At the time, researchers elsewhere had engineered a hepatitis B virus (HBV) vaccine into yeast cells. “I wondered whether it might be possible to produce a similar recombinant vaccine in plants,” Arntzen says. It was an unusual idea. “Some people dismissed the idea as totally unworkable,” Arntzen says. “They gave us a look like we were naïve plant molecular biologists who didn’t understand immunology.”

Still, Arntzen decided to try. His lab first chose tobacco, a well-understood plant, to test the plant vaccine idea. They spliced a gene encoding an HBV surface protein into Agrobacterium. Invading the tobacco cells, the Agrobacterium directed the cells to produce the HBV antigen. The researchers regenerated the cells into tobacco plants.

Inspecting electron micrographs of tobacco tissue extracts. Arntzen was thrilled at what he saw: tiny, viruslike particles of NBV. “It was incredible. To be honest, I almost expected the plants to degrade the antigen. Plants usually do that to nonuseful proteins. And here we come along, putting in a protein of a human pathogen… it was very exciting that it worked.”

Arntzen and colleagues next crushed the tobacco plants’ leaves, extracted the HBV antigen, and injected it into mice. Sure enough, the mice developed antibodies to the antigen.

Now that the lab knew the technology worked, they turned to a well-known food crop – be potato. Using their modified HBV antigen, the researchers added a DNA sequence that causes proteins to be expressed in potato tubers. They used the same process as before and achieved the same result: antibodies surfaced.

Although injecting plant-produced vaccines in mice is a good way to test the con? Researchers really want animals to gain immunity by eating a vaccine in food. So far, mice that eat HBV-tainted potatoes have not responded with significant antibody protection.

“We just don’t know yet if we can get [the response] with HBV through an oral route,” Arntzen says. On the other hand, the researchers have documented antibody response in mice feeding on potatoes laced with the E. coli toxin. Why the difference? It appears that the E. coli toxin, a potent immune system stimulator, is better than HBV at alerting immune cells secreted into the digestive system. This makes sense, given that the E. coli protein causes a digestive disorder.

The researches are also pursuing potato studies with Norwalk virus, which is responsible for about half the outbreaks of gastroenteritis in the United States. Mary Estes, a molecular virologist at Baylor College of Medicine in Houston, is a key collaborator in the Norwalk work.

“We had already produced the Norwalk virus capsid protein in another system [insect cells] and knew it self-assembled into particles which were immunogenic when given orally to animals,” Estes said. “So I was optimistic that we could make similar particles in plants, and this was a great idea.” At press time, the group’s first report showing that Norwalk virus expressed in potatoes is immunogenic had been submitted for publication.

For all its potential, the potato remains a somewhat impractical edible vaccine crop. “For one thing, you really need something that can be eaten raw,” says Gregory May, a plant biologist at Boyce Thompson. “If you cook the potato, its engineered proteins are going to denature and lose their function. And who eats raw potatoes?”

Enter the banana, a food almost always eaten raw. “In the developing world,” May says, “people eat bananas all the time. They make banana flour and banana beer. And they feed bananas to young children.”

Step by step, May and colleagues are engineering a vaccine-carrying banana. Last spring, the group reported its first milestone: they successfully engineered a marker gene into the fruit. They will soon attempt engineering vaccine antigens as well. The banana research involves collaboration with a research lab in Irapuato, Mexico, which has field facilities to grow the tropical fruit. “We would like to establish a collaboration with Mexico’s Ministry of Health,” Arntzen says. “They have a good infrastructure for public education about health issues.” In that country, he notes, diarrheal disease is the number one killer of children under five.

Technical Hurdles. Three years ago, no one had ever tried to engineer a vaccine-carrying plant. “We’re starting with near-zero information on inserting foreign pharmacological proteins in plants,” Arntzen says. “So we do a lot of empirical groundwork.” Much of that work involves boosting gene expression. Often, an engineered disease gene produces a protein in plant tissue, but at levels too low to generate an immune response if the plant were eaten.

“Gene expression is a continuing challenge,” notes Hein. One way researchers hope to maximize expression is to learn how some naturally occurring genes become highly expressed in the first place. “We’re trying to identify proteins present in abundance in the banana,” explains May. “Then we can backtrack to look for the regulatory elements that determine the proteins’ expression.” Researchers would then attach the DNA sequences that drive high gene expression to their disease antigen gene. Using this technique, the group increased E. coli protein expression in potatoes tenfold, Mason says.

Once researchers get enough of a disease protein into a plant, the next challenge is to make sure an animal’s immune system responds to that protein. One problem is oral tolerance. “Many antigens that you eat in food do not give an immune response,” Mason says. “That’s because the body perceives the food antigen as food, and the immune response is essentially repressed.”

To alert the immune system, the researchers may try to pair vaccine disease proteins with vary strong oral immunogens, like the cholera toxin or Norwalk virus. These adjuvants may help stimulate an immune response.

Meanwhile, a lot of questions remain. “They range all the way from kitchen science, like what happens to the protein when you crush alfalfa, dry it, and make it into food, to long-term immunization issues,” Hein says.

The next goal for plant vaccine engineers is to demonstrate that protective immunity is gained by eating an engineered plant. So far, the experiments with mice have shown only that they generate antibodies when presented with a disease antigen. These mice have never actually encountered natural E. coli or HBV diseases, because animals don’t get these human diseases.

The only way to truly test whether a plant vaccine confers protective immunity is to deliver that plant to a human, who is then exposed to the disease under investigation. “This is the acid test,” says Mason. “In some cases, an antigen preparation may stimulate immunoglobulins that neutralize the antigen in the test tube and yet provide little or no protection against the disease. We are planning to begin some human studies with the potatoes, initially only to assess safety and immunogenicity in humans, but later to assess protection.”

Clinical trials should answer a host of questions, including whether normal plant proteins interfere with disease antigen delivery or how well various immunizing proteins act when taken orally. Clements, for one, is confident the plant vaccine technology will prevail. “It’s going to take some time to develop,” he says. “But there sure is a lot of potential.”

Check Yourself

Ex. 1 Choose the correct word corresponding to the following definitions

parasite a) Body – building substance essential to good

health, in such food as milk, eggs, meat.

microscopic b) Substance used in, or obtained by chemistry.

protein c) A group of microscopic, onecelled fungus plants.

chromosome d) Organical chemical substance (a catalyst)

formed in living cells, able to cause changes in

other substances without being changed itself.

chemical e) Animal or plant living on or in another and

getting its food from it.

bacteria f) A microorganism that is capable of growing in the

complete absence of oxygen.

fungus g) Plant without leaves, flowers or green coloring

matter, growing on other plants or on decaying

matter.

virus h) Microscopic unit of living matter enclosing

a nucleus with self – producing genes.

cell i) Any of various poisonous elements, smaller than

bacteria, causing the spread of infectious disease.

pathogen j) A scientific instrument that makes extremely small

things look larger.

prokaryote: k) Organism with a simple DNA chromosome

without a nuclear membrane and possessing a

small range of organelles.

enzyme l) A microorganism associated with disease.

anaerobe m) One of the minute threads in every nucleus

in animal and plant cells, carrying genes.

Ex. 2 Fill in the blanks with the suitable word

bacterium / strain / microbiology / cell / viruses / organisms / spiral / bacteria / origin / diseases / microscope / diploccocus

To see microbes, you need a ………….. .

………….. can destroy the …………. they invade.

Microbes are small ............... , generally smaller than human eyes can detect.

The …………… Borrelia burydorferi has …………… form.

…………… of eukaryotes remains obscure and speculatic.

Infectious .………… have played major roles in shaping human history.

Industrial ………….. came of age in the 20 th century.

..................is a pair of cocci.

Some …………. help make pharmaceuticals.

…………..is a group of organisms of the same species, having distinctive characteristics but not usually considered a separate breed or variety.

Ex. 3 In each of the following sentences there is one mistake. Find it and

correct it

1. How microbes are small?

2. Microbes can live as a single sell.

3. We have knowed this disease for ages.

4. He reads the books about enzymes at that moment.

5. They by fission reproduce.

6. Where viruses act?

7. Many protozoans prey on soil bacteria.

8. What does lead to disease?

9. A virus lives inside a houst cell?

10. Students examined molds under the microscope from 9 to 10 yesterday.

Ex. 4 Make up questions to the following statements

1. That filter stopped ordinary bacteria.

2. Human immunodeficiency virus can damage organs directly.

3. The exact nature of the viruses has not yet been settled.

4. Several types of microbes will be seen.

5. The scientists want some bacteria to make pesticides.

6. A virus that causes yellow fever in man was found in 1901.

7. Rickettsia are larger than viruses.

8. The largest known virus measures 450 millimicrons.

9. Bacteria are being put to work in wondrous ways.

10. Some biochemical examination should be carried out.

Ex. 5 Translate into English

- Отличительные признаки микроорганизмов - ничтожно малые размеры и простота биологической организации.

- Микроорганизмы легко переносятся потоками воздуха, насекомыми и птицами на значительные расстояния.

- Важная особенность микроорганизмов – их способность приспосабливаться к изменяющимся условиям окружающей среды.

- Морфология микробов – внешняя форма и внутреннее строений.

- В настоящее время внутреннее строение микробных тел успешно изучается при помощи электронной микроскопии, посредством чрезвычайно тонких срезов бактерий.

- Величина микробов измеряется в микронах. Вес микробов так мал, что 100 млрд. бактерий весит 1 гр.

- Химический состав микробов: тела микроорганизмов содержат те же химические вещества, которые находятся и у высших растений и животных. Это говорит о материальном единстве и физиологическом родстве всего живого мира. На всех уровнях строения живых организмов в их состав обязательно входят 16 важнейших элементов: C, O, N, H, P, S, Fe, Cu, Na, K, Ca, Mg, Co, Ce, I, Mn. Их называют элементами жизни.

- Большая часть одноклеточных микробов относится к группе бактерий. Эта группа весьма широко распространена в природе; обычно представлена или маленькими кокком (шариком) или маленькой палочкой (прямой или изогнутой). У бактерий выделяют три основные формы клетки: сферическую, цилиндрическую и извитую. Сферические, или шаровидные бактерии называются кокками (от греч. “coccus” – ягода). Различают: а) монококки,

б) диплококки, в) стрептококки, г) тетракокки, д) сарцины, е) стафилококки.

- Жизнь микроорганизмов неразрывно связана с окружающей средой. С одной стороны, деятельность микробов значительно изменяет окружающую среду в результате удаления из нее питательных веществ и выделения продуктов обмена; с другой стороны, интенсивность обменных процессов внутри клетки во многом зависит от условий окружающей среды. К важнейшим физическим факторам, обуславливающим активность микроорганизмов, относятся температура и свет.

- Микроорганизмы являются наиболее простыми формами жизни. Поэтому они представляют собой весьма удобные модели для изучения многих проблем общей биологии, выяснения сущности явления жизни, овладения и управления жизненными процессами, в частности обменом веществ и наследственностью организмов. В настоящее время биологические науки не могут развиваться без изучения генетики, в частности, генетики микроорганизмов.

- Наша страна является родиной науки о вирусах, а Д.И. Ивановский справедливо считается основоположником этой науки. К настоящему времени открыто большое количество вирусов человека и животных (около 300), растений (более 200) и продолжают открывать все новые вирусы. Вирусы являются самыми мелкими из всех микроорганизмов. По внешнему виду одни вирусы имеют шарообразную форму (вирус гриппа), другие кубовидную форму (вирус оспы), третьи вид палочки.

- По вопросу о происхождении вирусов имеются две гипотезы. По одной гипотезе и вирусы, и фаги являются регрессивными формами одноклеточных организмов, утративших клеточную структуру в результате длительного внутриклеточного паразитирования, в организме животных и растений. Большинство исследователей придерживаются другой гипотезы, согласно которой современные вирусы и фаги являются потомками первичных возникших на Земле доклеточных организмов.

- Микробиология в той или иной степени связана с др. науками: морфологией и систематикой низших растений и животных, физиологией растений, биохимией, биофизикой, генетикой, эволюционным учением, молекулярной биологией, органической химией, агрохимией, почвоведением, биогеохимией, гидробиологией, химической и микробиологической технологией и др. Микроорганизмы служат излюбленными объектами исследований при решении общих вопросов биохимии и генетики. Между микробиологией и химией существует постоянная конкуренция при выборе наиболее экономичных путей синтеза различных органических веществ. Ряд веществ, которые ранее получали микробиологическим путём, теперь производят на основе чисто химического синтеза (этиловый и бутиловый спирты, ацетон, метионин, антибиотик левомицетин и др.). Некоторые синтезы осуществляют как химическим, так и микробиологическим путём (витамин B₂, лизин и др.). В ряде производств сочетают микробиологические и химические методы (пенициллин, стероидные гормоны, витамин С и др.). Наконец, есть продукты и препараты, которые пока могут быть получены только путём микробиологического синтеза (многие антибиотики сложного строения, ферменты, липиды, кормовой белок и т.д.).