Viruses Lesson 3 PDF

**Viruses---Borrowed Life** Viruses fail to fulfill the classic criteria for a living organism, i.e., they have **no metabolism of their own** and they use the reproductive mechanism of their host animals, plants, or bacteria for their own reproduction. This inability to reproduce outside host cell...

**Viruses---Borrowed Life** Viruses fail to fulfill the classic criteria for a living organism, i.e., they have **no metabolism of their own** and they use the reproductive mechanism of their host animals, plants, or bacteria for their own reproduction. This inability to reproduce outside host cells makes it impossible for them to survive independently. If they are not living organisms, what are they? They are basically **programs** that are added into the genome of their hosts, hijacking their reproductive mechanisms to produce more viruses---very much like computer viruses. Because viruses use the hosts' metabolic functions, inhibitors such as **antibiotics have no effect on them**---there is no metabolism to be disrupted. The only point of possible intervention is viral interaction with the host. There are two kinds of viruses---**enveloped viruses and naked viruses**. The genome of naked viruses is protected by a **capsid** (Engl. *core*), whereas **enveloped viruses** form their envelope by pinching off a bud from the host's cell membrane, into which they insert virus-coded proteins. This lipid--protein viral envelope contains a capsid, similar to that of naked viruses. Unlike microorganisms, viruses **only require their nucleic acid and a host cell** for their reproduction. Some viruses, however, **also contain the enzymes** they need for replication. This applies to retroviruses that contain **reverse transcriptase** in their capsid. Viruses are **completely dependent on their host cells** as far as the reproduction of their nucleic acid and the synthesis of their proteins is concerned. Some viruses reproduce using the **lytic cycle** (Greek *lysis*, dissolution---a word that also gave the name to Fleming's lysozyme, Chapter: Enzymes: Molecular Supercatalysts for Use at Home and in Industry), during which the virus is released and destroys the host cell. Other viruses prefer a **nonlytic cycle** in which they form buds that are pinched off from the cell membrane (this is how it works in enveloped viruses such as influenza viruses or HIV). All viruses contain a single type of nucleic acid: RNA or DNA. **They are classified according to their nucleic acid**, their protein covers, and their host specificity. Examples of **RNA viruses** include the AIDS virus HIV, the influenza viruses, the measles virus, the rabies virus, and a plant virus known as tobacco mosaic virus (TMV, see Chapter: The Wonders of Gene Technology). The latter two are rod-shaped. Other viruses in this category include the picornavirus group, e.g., poliovirus and rhinovirus, which is responsible for colds, and the Ebola virus that has caused so many deaths in Africa. The **SARS** (severe acute respiratory syndrome) **virus** is a major cause for concern, especially in Hong Kong, China and Canada. This is another RNA virus, called a **coronavirus**, because its surface resembles a crown (*corona*, Latin for crown). **DNA viruses** include, e.g., papovaviruses, which mostly cause warts, but some species may cause tumors. Others are ***Variola* (smallpox)** and ***Vaccinia* (cowpox)** viruses, **herpes** viruses, **adenoviruses** (causing infections of the mucous membrane), **bacteriophages** that attack bacteria (Greek *phagein*, to eat), and **baculoviruses** that exclusively attack insects. **5.2 How Viruses Attack Cells** Viruses always bind first to the surfaces of cells. DNA viruses such as **bacteriophages** inject their genetic material (double-stranded DNA) into the bacterial cel. With the help of the bacterial cell, they produce enzymes (DNA and RNA polymerase) that are used to synthesize DNA and mRNA. The viral mRNA is synthesized by the bacterial RNA polymerase and read by the bacterial ribosome. Thus, the bacterial cell produces the viral protein envelope as well as its DNA from bacterial building blocks. Eventually, the parts that make up a bacteriophage assemble join to form a **complete bacteriophage that lyses the host cell**. In some cases, however, viral DNA is inserted into bacterial DNA **without lyzing** the cell. Such DNA is called **dormant viral DNA**, which will only be released and reproduced in later bacterial generations. In animal cells, viruses bind to receptors on the cell surface, and the protein envelope merges with the cell membrane to let the virus enter. In **RNA viruses** of the retrovirus group (e.g., HIV), single-stranded RNA enters the cell and is converted into double-stranded DNA with the help of an enzyme carried by the virus (**reverse transcriptase**, Chapter: The Wonders of Gene Technology). The transcribed viral DNA is inserted into chromosomal DNA in the nucleus. The transcription mechanism of the host cell (RNA polymerase) then transcribes it into mRNA, which becomes a blueprint for the synthesis of viral proteins in the ribosomes. These include nonstructural proteins which are the cause of the pathogenicity of many viruses. The newly produced viral RNA and the viral proteins assemble to form new viruses which exit the cell. Only **very few types of viruses integrate their genome** into that of the host. These include herpes viruses and retroviruses. Genome integration enables a virus to remain stable in the genome of the host cell over many generations of cell division. The **infection is dormant** and the infected organism may display absolutely no sign of any disease or malfunction. In other cases, viruses such as the hepatitis B virus or some papilloma viruses cause DNA breakages when integrating host cell genomes. Such **"abortive integration"** is partly responsible for the **development of tumors**. For example, nearly 80% of all cervical cancers are caused by specific variants of HPV (human papilloma virus). The search for **strategies against the attack of viruses** is a major undertaking (Box 5.1). It is conceivable that specific **antibodies** (see later in the Chapter) could trap and neutralize viruses before they dock onto the host cell and invade it. Antibodies could also prevent invasion by **masking the relevant binding sites on the target cells** so that the viruses would fail to recognize them (Fig. 5.9). Antibodies can also **tag viruses** so that they can be recognized and destroyed by immune system cells, such as **macrophages** and granulocytes. Retroviruses can be fought **with reverse transcriptase inhibitors** (see Chapter: The Wonders of Gene Technology) that prevent the transformation of RNA into DNA. Another approach is to use **antisense RNA---**an exact mirror image (see Chapter: Analytical Biotechnology and the Human Genome) of the viral RNA---which it can bind and inactivate (Box 5.1). A fairly recent strategy consists of employing **short double-stranded RNA sections** (**RNAi**---the *i* standing for interference. More details in Chapter: Myocardial Infarction, Cancer, and Stem Cells: Biotechnology Is a Life Saver). Artificially created RNAi, between 21 and 23 nucleotides long, was used by German scientist **Tom Tuschl** to silence mammalian genes without triggering a disruptive interferon response (which would have led to the degradation of all RNA present). Since then, it has been possible to silence specific genes---e.g., for HIV, the *nef, rev, gag,* and *pol* genes. There are also initial successes in the fight against influenza and hepatitis C viruses. Many HIV therapeutics are based on **inhibitors of virus-coded protease**, which plays an important part in the maturation process of viral proteins. All these different routes are followed in contemporary AIDS research (Box 5.1). As the **HIV virus mainly attacks T-helper cells** that control the human immune response, it should be possible to strengthen the immune response by providing **genetically engineered cytokines: single molecules such as interleukin-2 that modulate immune responses**. In a first step, the virus could be put out of action with "chemical weapons," and then immune cells could be stimulated by interleukin-2. Some viruses cause infected cells to produce another type of cytokines: **interferons**. The secreted or artificially introduced interferon binds to specific receptor molecules on the surface of other, uninfected cells, making them resistant to the virus. Just like the lymphokine **interleukin-2** (**IL-2**), interferons were first enthusiastically hailed as the **wonder drugs of the future** that would be able to cure a host of diseases from the common cold to cancer. However, they did not fulfill these unrealistic expectations. Their effectiveness as a cure is limited, whereas the side effects are often considerable. However, like IL-2, they have their place in the treatment of human disease, usually in combination with other medications (see Chapter: Myocardial Infarction, Cancer, and Stem Cells: Biotechnology Is a Life Saver) **How the Body Defends Itself Against Infections---Humoral Immune Response Through Antibodies** When Europeans conquered the Americas, they were assisted by **biological weapons** of which they were not aware at the time---bacteria and viruses. These killed a large proportion of the native inhabitants. Between the 16th and the 19th century, the influx of **conquerors and settlers into the Americas and Oceania brought measles, smallpox, influenza, typhoid, diphtheria, malaria, mumps, whooping cough, the plague, tuberculosis, and yellow fever**, whereas the natives had just one fatal pathogen "on their side"---syphilis. It seems that epidemics were something unknown on the American continent. According to **Jared Diamond**, the inequality of weapons between Red Indians or Indios and Europeans was caused by the presence of the **large cattle herds** of the settled Eurasian farmers. These herds became the breeding ground for acute and endemic diseases that later spread to similarly crowded human settlements. This explains why outbreaks of infectious diseases as we know them are a fairly recent phenomenon. Smallpox appeared for the first time in 1600 BC, mumps and the plague at 400 BC, and cholera and louse-borne epidemic typhoid only in the 16th century. Over several hundred years, the **Eurasian population** had been able to develop **immunity** to the diseases and reached a stalemate. The inhabitants of the New World, by contrast, did not have time to arm their totally unprepared immune systems and were almost completely wiped out by the pathogens. **How does the immune system protect us**? Let us just resort to a simplified answer at the moment, as the immune system is so complex that a full explanation would go beyond an introduction to biotechnology. The immune system distinguishes between **"self" and "nonself"** and has the ability to produce a **hundred million (108) different antibody specificities** and over a **trillion (1012) different T-cell receptors**. It consists of two closely interconnected systems with parallel actions---the humoral and the cellular immune response. The **humoral immune response** (Latin: *humor*=liquid) uses soluble proteins, **antibodies** also known as immune-globulins (Box 5.3), as recognition elements. There are also humoral defense factors, including lysozyme (see Chapter: Enzymes: Molecular Supercatalysts for Use at Home and in Industry), interferons, and other cytokines. Antibodies bind to foreign molecules or cells, thus labeling them as intruders and encouraging phagocytosis by macrophages. Antibodies are produced by plasma cells, which, in turn, are derived from B-cells. **B-cells** or **B-Lymphocytes** derived their name from *Bursa fabricii*---a lymphatic organ that is unique to birds which lies in the end section of the cloaca (Fig. 5.13). Lymphocytes develop into **B-lymphocytes** in that bursa. If the bursa were removed from a chicken, it would become very susceptible to bacterial infections and would be unable to produce antibodies. A **foreign macromolecule (or a cell or virus)** that elicits an immune response is called an **antigen**. Antibodies do not target the whole antigen, but only a portion of it, which is known as **epitope** or **antigenic determinant**. An **infection** mobilizes several cooperating immune cell populations. **B-lymphocytes** carry antibodies as recognition molecules on their surfaces (membranebound immunoglobulins). However, they are usually not activated by circulating antigens. These are taken up by **antigen-presenting cells**---either **macrophages** or **dendritic cells**. They process the antigen, move antigen fragments to the surface and present it to T-helper cells. The fragments on these antigen-presenting cells act as a stimulus on the T-cells to produce interleukin-2, which, in turn, activates the B-cells that have also been in contact with the antigen. These begin to proliferate and form a cell clone (**clonal selection**). Some of the resulting daughter cells become **memory cells**, which ensure a rapid immune response in the case of reinfection, while others develop into antibody-producing plasma cells. The freely circulating antibodies bind to the antigen and cells that may carry it, thus **labeling the enemy for destruction by** other components of the immune system. These mechanisms are supported by the body's own **complement system**, a cascade involving about 30 proteins, and by ADCC (antibody-dependent cell-mediated cytotoxicity), the destruction of foreign or abnormal cells by specific immune system killer cells. Whether dissolved in the blood plasma or cell-bound, both the complement and ADCC systems form a defense line against microorganisms (e.g., bacteria, fungi, or parasites). Because of their powerful celldestroying properties, they can cause tissue damage when not properly regulated, which may occur due to various diseases (heart attack, systemic *Lupus erythematodes* or rheumatoid arthritis). *How viruses attack cells. Left: bacteriophages attack Escherichia coli. Right: A retrovirus* *attacks a human cell.* *T cell receptor*   **The First Vaccination: Cowpox Against Smallpox** If the English country doctor **Edward Jenner** (1749-- 1823) were to repeat his famous experiment of 1796 today, he would soon find himself in prison. He injected 8-year-old **James Phipps** with a sample from a cowpox pustule of dairy girl **Sarah Nelmes** (Fig. 5.15). Then, 2 months later, he injected the boy with a potentially lethal dose of smallpox. This would nowadays be a violation of even the most lax safety guidelines, only exceeded by the experiments of **Louis Pasteur** (Box 5.5). However, the boy survived, and Jenner helped to revolutionize medicine. The **first vaccine** (Latin *vacca*, cow) in history had been found, although it has been said that there were already active smallpox vaccinations as early as 1000 BC. Nowadays, biotechnology is revolutionizing vaccination---making it possible to develop new vaccines in a very short time while dramatically reducing the risk involved. Jenner had correctly observed that overcoming cowpox conveyed lifelong immunity in humans, not only to cowpox, but also to smallpox. What Jenner did not know was that the cowpox virus is closely related to the smallpox virus. As explained above, in case of infection by either virus, lymphocytes in the blood can trigger an alarm at the intrusion of antigens, causing on a command to other cells to produce antibodies. Antibodies, in turn, tag the pathogens for macrophages to destroy virus-infected cells and viral particles. As mentioned above, B-cells proliferate after antigen contact and activation by T-helper cells. While a proportion of their offspring produce large quantities of antibodies to tag the intruders, some become **memory cells** that enable a fast immune reaction, should the organism be reinfected in the future. Memory B-cells can remain in the system for life---conveying **lifelong immunity** to the relevant antigen. **Jenner was in luck** because the **similarity in the structure** of **cowpox and smallpox** ensured that immunity was acquired not only for the innocuous cowpox, but also for the far more dangerous smallpox. It is thus possible to prepare the immune system for the attack of lifethreatening pathogens by exposing it to innocuous ones. The last smallpox patient in the world, the Somali **Ali Maow Maalin**, was discharged from hospital on October 26, 1977. Over the following 2 years, the world population was scrutinized for smallpox and **finally declared smallpox-free.** Only two laboratories in the world still store stocks of smallpox viruses (one hopes!)---the international WHO **reference labs in Atlanta (United States) and near Novosibirsk (Russia)**. There has been a debate about the wisdom of keeping the remaining stocks. In view of possible **bioterrorist** attacks, however, all industrial nations keep a stock of smallpox vaccines. Who would be confident enough to say that smallpox viruses will not get into the wrong hands? It may already have happened. At the beginning of the 19th century, half a million people per year contracted smallpox in Germany alone. One in ten died, and faces scarred by smallpox were a common sight. But this sad fact belongs to the past. **For the first time in history, a disease has been eradicated due to vaccination.** Unfortunately, there are not many pathogens that have such close innocuous relations, and only with **Louis Pasteur**, born the year before Jenner died, did the systematic search for vaccines begin.   **\ ** **Contemporary Vaccination** Nowadays, vaccination relies on toxoids for vaccination, killed or weakened live pathogens, and antigens produced by recombinant DNA technology. Thanks to the success of genetic engineering, research is also being done on modified live vaccines and peptide vaccines (Figs. 5.18--5.20). **Toxoids** are extracts from toxins released by pathogens. They are neutralized (sometimes using formalin) but can still stimulate the body's immune system, when injected. Vaccines against tetanus and diphtheria belong to this class (Fig. 5.21). The **tetanus pathogen *Clostridium tetani*** (the one who dwells in the ground), e.g., can infect an open wound and inject a neurotoxic protein into the bloodstream. This results in spastic paralysis---it used to be a shocking sight among soldiers wounded in battle. The **tetanus vaccine** consists of an inactivated neurotoxin, requiring a booster every 10 years to maintain a sufficient number of antibodies circulating in the system. Cholera, polio, and typhoid vaccines consist of chemically **killed bacteria or viruses**. In other words, these vaccines contain pathogens that cannot cause the disease, but that retain all the antigens. **Cholera vaccine**, e.g., cannot cause an outbreak of the disease, even though it contains the cholera bacterium toxin (it has been rendered ineffective). Cholera vaccine is simply swallowed: it is an **active oral vaccine**. It is said to be active because the organism produces its own antibodies against the killed bacteria and the toxin. Cholera vaccination provides strong protection against the disease: approximately 90%. Adults and children from 6 years on are given two vaccinations, between 1 and 6 weeks apart. Protection begins 8 days after the vaccination and lasts about 2 years. **Rubella and measles vaccinations** rely on **attenuated** (weakened) **pathogens.** Unfortunately, there have been a number of incidents in which the pathogens had not been sufficiently attenuated. Various **genetically engineered vaccines** for humans and animals have been in use since 1985, e.g., against foot-and-mouth disease in cattle. A recombinant vaccine has been recently developed against strains of HPVs that cause the majority (80%) of all cervical cancers. How effective these vaccines truly are against preventing cancer is not yet known. The first genetically engineered vaccine designed for humans was approved in 1986 in the United States. It protects against **hepatitis B,** a chronic disease caused by a DNA virus (HBV) that affects some 240 million people. 686,000 people die every year due to hepatitis B. It causes one of the most frequent infectious diseases worldwide, alongside tuberculosis and HIV. Up to 25% of people affected die from HBV sequels that include cirrhosis or carcinoma of the liver. The virus is endemic in South East Asia and in sub-Saharan Africa. Thanks to vaccination programs, its presence in the Western European and US populations has been reduced to 0.1% chronic virus carriers. The conventional production of hepatitis B vaccine was facing huge problems. In contrast to most other microorganisms, **hepatitis B viruses cannot be bred in conventional nutrient media or animal embryos** (such as fertilized chicken eggs). Infected blood had to be used instead. The vaccine manufacturing process consisted of isolating viruses from the blood of infected carriers, detaching viral envelope proteins with detergents (which mostly destroy the viruses), and purifying them. These envelope proteins provoked immune reactions and thus made the vaccines. Infected blood is, of course, dangerous to work with. **All members of the lab had to be immunized**, i.e., vaccinated, and the work was carried out in isolated secure labs. The matter was further complicated because each batch had to be tested on chimpanzees (of which, for ethical reasons, there were only a limited number available) in order to make sure no live virus remained. A full year was needed to produce a batch of hepatitis B vaccine in this way. Unsurprisingly, only a very limited amount of natural vaccine was available and only highrisk groups could be vaccinated. The new **genetically engineered vaccine** against hepatitis is produced by genetically modified eukaryotic cells in culture: yeasts or mammalian cells. Both types of cells produce a viral surface protein. Because no virus is present at any stage, the new vaccine can never cause hepatitis. Vaccine production in *E. coli* has not proved to be very effective because bacteria are not able to imitate the protein modifications carried out by pathogens, such as glycosylations in particular (see end of Chapter: The Wonders of Gene Technology). DNA vaccines constitute a possible alternative to vaccines against proteins. The idea here is to introduce an individual gene that encodes an antigen and express it inside the organism. Making a DNA vaccine would be very simple. Unfortunately, this approach is limited because of unwanted immune reactions, i.e., allergies. **Recombinant vaccines** represent a major breakthrough in the field. They can be manufactured whenever a surface protein of a pathogen can be shown to cause an immune response. The gene responsible for this protein is isolated from the pathogen's genome and inserted it into that of harmless microorganisms such as bakers' yeast or mammalian organisms, which could then produce large amounts of the protein. There is an added bonus to the method: viral contamination of the vaccine is impossible. The most comprehensive and disastrous vaccination program ever carried out in Germany started in late October 2009, after the outbreak of the **swine flu virus**. Little information was available about the vaccine's benefits and risks and the public opinion about mass vaccination was hesitant and skeptical. In late 2011, the *Pandemrix* vaccine reached its expiration date. Some 16 million doses, worth 130 million Euros, were burned at a temperature of 1000°C, contributing got one of the greatest flops in the history of German health services. **Live Vaccines** Rabies has been an almost worldwide scourge for most of history, with the exception of North Western Europe, Japan, Australia, and a few Pacific islands. But nowadays, foxes in Europe's woodlands are biotechnologically protected. Baits are laced with **live vaccines**. In live vaccines, innocuous viruses such as the cowpox virus (*Vaccinia*) are used as vectors to transport foreign genes. genetically engineered versions of three HIV genes. The HIV proteins that are expressed pose no risk whatsoever, but elicit an immune reaction in the organism. **Edible vaccines** are highly controversial. In Chapter 7, Green Biotechnology, we will look at the creation of **transgenic plants**. Specifically marked (perhaps through blue pigment, bananas or potatoes could be us ed to produce such oral vaccines and be ingested as food, the antigen traversing the gastrointestinal tract and conferring immunization. **Recombinant Antibodies** Before the emergence of gene technology, the traditional way of obtaining antibodies was by immunization (**polyclonal antibodies** isolated directly from animal serum) or by hybridoma technology (**monoclonal antibodie**s). These techniques are laborintensive and imply animal research. Nowadays, it is possible to produce antibodies without animals, using only cell cultures and viruses. The technique yields the antigen-binding fragments (**Fab fragments)** of antibodies, the very regions responsible for the endless variety of binding specificities. Traditionally, Fab fragments could only be obtained from complete antibodies by cleavage with proteases. The procedure has now been replaced by a smart recombinant DNA method. Recombinant antibodies are also monoclonal (derived from a single clone) and highly specific, binding precisely to their epitope. The crucial sites for antibody binding are the variable regions (Fv). They lack the stabilizing disulfide bridges of constant chains and need extra stabilization. This is usually done by chaining peptides that turn them into single protein strands. They are also elongated by a tag that modifies theirbiochemical or surface-binding properties. This results in **single-chain Fv fragments** (scFv fragments). Why produce **recombinant antibodies**, since the immune system can do so on its own? For one thing, the absence of animal involvement should please animal lovers. But it also offers better safety, since animals can transmit viruses that are potentially dangerous to humans. The production of antibodies using *E. coli* cells is also **vastly cheaper and faster** than production in hybridoma cells. Finally, recombinant antibodies rely on the entire range of molecular biology techniques developed for *E. coli*, offering unsurpassed ease and biotech power! **Recombinant Antibody Libraries** Even a very successful fusion of myeloma (cancer) and spleen cells into hybridoma cells can only yield a few dozen different antibodies---a hundred at the most. This is not terribly impressive, keeping in mind that our own **immune system is capable of producing 100,000 antibodies with different specificities**. How could we tap into this vast potential? Recombinant antibody generation techniques bypass the ineffective hybridoma-generating fusion step. After immunization of an animal with an antigen, spleen cells are collected, and their mature mRNA is isolated (see Chapter: The Wonders of Gene Technology). Using reverse transcriptase, the singlestranded mRNA is copied into doublestranded copy DNA (cDNA). PCR (see Chapter: Analytical Biotechnology and the Human Genome) is then used to produce millions of copies of cDNA encoding the light and heavy antibody chains (Fig. 5.31). The cDNA copies are cut with restriction endonucleases, resulting in fragments with sticky ends, which are then cloned into bacteriophage λ- derived vectors. Two different **libraries** are created: one with DNA encoding the heavy chains (**H-chains**) and the other with DNA encoding the light chains (**L-chains**). The two phage libraries are then combined with a third helper **phage**, and the mixture is used to infect a lawn of *E.coli* cells growing in a Petri dish. Infection with all three phages creates recombinant phage particles that lyze *E. coli* cells, yielding plaques ("puddles") containing billions of phages. If recombination brings matching DNA sequences for H and L chains in the same phage, the plaque will also contain **Fab fragments.** To find such phages, an imprint of the plaques is made on a nitrocellulose filter. Like all proteins, Fab fragments bind firmly to the filter. After adding a radioactively labeled antigen and washing out unbound antigen, the filter is put on X-ray film. Black spots of the film will show which plaques contain binding Fab fragments and the phages that made them! Once the plaques are identified, Fab-encoding DNA is isolated so that it can be inserted into bacterial or mammalian vectors. This increases the availability of possible antibodies at least by a 1000-fold, compared to standard hybridoma technology. **5.12 Piggyback or Phage Display** The B-lymphocytes needed by the immune system are selected **thanks to a membrane-bound antibody on their surface** (see above). An antigen (e.g., of a virus) that binds to it and IL-2, produced by T-helper cells, stimulate the B-lymphocytes to divide (clonal selection). The **antibody indicates the "street number"** of the relevant gene that produces it in its parent lymphocyte. In other words, the antibody carries its gene piggyback, as if in a huge backpack (Fig. 5.32). It is easy to identify the B-lymphocytes with the desired antibody because it makes a clone. But could we do the same in bacteria by reading the "street number" in a simpler way, to fish out the right gene? That would be a genetic engineer's dream! This dream was turned into reality by **George P. Smith** at the University of Missouri in Columbia, Missouri, United States, in 1985. He solved the problem not using bacteria, but **bacteriophage M13**. Unlike phage λ (see Chapter: The Wonders of Gene Technology), it is a filamentous phage, and with a much smaller genome. It consists of circular single-stranded DNA, containing only a few genes for capsid proteins and for a simple infection cycle (Box 5.10). The cunning virus enters *E. coli* via its sex pili, introducing its single-stranded DNA through an F-pilus. The DNA acts as a template to form double-stranded DNA in the cell, which is not inserted into the bacterial genome, but replicated between 100 and 200 times. When the bacterium divides, each daughter cell receives multiple copies of the phage DNA. M13 is considerate enough not to kill its host and only to slow down its growth. After single-stranded DNA copies have been made and packed into filamentous protein envelopes, the phage particles are released. A tubule is formed by 2700 type pVIII proteins. Most remarkable, however, are the five proteins of type pVII and pIX at one end and pIII and pIV at the other end of the phage (Fig. 5.33). This remarkable feature is that **pIII remains functional** even after integration of foreign sequences. Smith hypothesized that if a foreign gene were inserted into the *pIII* gene of the phage, it should show up as a foreign protein on the phage envelope, on its surface. And it worked! This method, called **phage display** was first used to find the gene for a strongly binding growth hormone. **Ongoing Hope for Cancer Patients---Antibody Targeted Therapies** "Imagine a new cancer treatment, based on the cruise missiles principle. A submicroscopic rocket with an automatic search head is launched in the body through injection. It searches out cancer cells and destroys them without attacking normal healthy tissue. Such a wonder weapon does not exist yet, but there are indications that it could be available in the near future." These words were published in 1981, in a newspaper that is definitely not prone to hype, namely the *Wall Street Journal.* The author of this prophetic article did not exaggerate: the advent of monoclonal antibodies has revolutionized the treatment of many cancers. Today, more than a dozen approved anticancer MAbs are benefitting millions of patients worldwide, and several hundred are at various stages of development. All anticancer monoclonal antibodies abide one fundamental principle, called **targeted therapy**. As in tumor diagnostics, therapeutic antibodies recognize cell-surface antigens that differentiate cancer cells from their healthy counterparts. But they do much more than simply tag cancer cells: they inhibit their growth and may even promote their death via multiple mechanisms. Thus, by binding well-chosen membrane proteins, monoclonal antibodies can target key mechanisms that make the cells cancerous (hence the term **targeted therapy**). HER2 is an example of an **oncogene** (a mutated gene that promotes cancer). Strong dependence on **oncogenes** exhibited by some cancers is called **oncogene addiction**. And just like with a drug addict, blocking the addiction has drastic consequences! The first generation of therapeutic antibodies are **chimeric** in nature, with two-thirds made up of human protein sequences (the constant region) and one third made of mouse sequences (the Fab region). The presence of animal sequences is a potential concern for unwanted side effects; subsequent antibodies were engineered to contain less and less nonhuman components. Second generation therapeutic antibodies are **humanized:** they contain less than 10% of nonhuman sequences (in the antigen binding region). Third generation therapeutic antibodies are said to be **fully human** because only human genes have been employed to build them (either in humanized mice or through phage display technologies). Chimeric antibodies are usually well-tolerated; humanized antibodies have fewer side effects, and human antibodies should be the safest of all.. **Paul Ehrlich**'s name for an antibody was the **magic bullet**, a name emphasizing its selective toxicity for certain pathogens. The first magic bullet was a chemotherapeutic drug called salvarsan and is known in the English-speaking world as arsphenamine. It was developed in 1909 by **Paul Ehrlich** and **Sahachiro Hata**. The German name is derived from Latin *salvare---*to save, *sanus*, healthy*---*and arsenic and could be interpreted as "healing arsenic." It is a milestone in drug research, as for the first time, a targeted antimicrobial drug was available to tackle a dangerous infectious disease. The effectiveness of arsphenamine was such that some infections like **syphilis** could be cured with a single injection.