ALS is a fatal motor neuron disease, and even though there are many different articles out there on the topic "How does ALS actually kill you?", none of them really delve deep enough into biology to adequately answer the question.
For instance, many articles cite "respiratory failure", or "CO2 poisoning". But the last time I checked, ALS doesn't make your lungs explode or cause your body to start producing toxic levels of CO2. So obviously, we need another level of detail here to support these claims. I'd be willing to be that with "respiratory failure", either the neurons innervating the diaphragm break down (hence you become physically unable to draw air in and push CO2 out), or perhaps the tiny/micronic structures inside your alveoli become impaired for some reason. If the former, then why wouldn't a ventilator/respirator help the patient?
Either way, I'm interested in a biological/neurological explanation for why ALS is considered to be (ultimately) fatal.
TL;DR Ventilation assistance does help but there are other factors.
ALS is a degeneration of the motor neurons, replacing them with hard material (i.e. sclerosis). The neurons innervating the muscles involved in respiration deteriorate and so it becomes difficult to breath, swallow, cough, etc. There's a compounding effect: we know that blood O2 and CO2 levels are balanced by respiration, O2 in/CO2 out, and we know that swallowing/coughing clear secretions from the respiratory tract. Basically you end up with trouble oxygenating your blood and riding it of CO2, hence CO2 poisoning. This can result in what is known as type 2 respiratory failure, characterized by low oxygen and high carbon dioxide levels (hypoxemia and hypercapnia). Low secretion clearance in the lungs can also result in aspiration pneumonia in addition to poor gas exchange.
Ventilation methods such as BCV are capable of mechanically assisting breathing, and maintaining ventilation-perfusion state in a way that prevents respiratory failure, but unfortunately doesn't slow the progression of the disease. The problem then is that in, for example, particularly severe cases, the quality of life has to be taken into account because tongue movement, gross movement in general due to motor neuron degeneration just fails. This can lead to what is essentially locked-in syndrome (LIS), and clinical outcomes can widely vary, though in this article they report the more pleasant outcomes of patients experiencing LIS. I think that for these cases due to poor communicaton patient-reported outcomes are fairly underreported, but family and caregivers at this point have to take into serious consideration options such as euthanasia. Cutting off ventilation can result in type 1 or 2 respiratory failure.
Now, I lack actual data for this statement but I should also point out that not all patients receive the same care or opt for assisted ventilation, in which case late-stage ALS would invariably lead to total neurodegeneration to breathing muscle innervation, where assisted ventilation becomes a requirement for survival.
Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig's disease or motor neuron disease, is a progressive, degenerative disease that destroys the nerve cells that control voluntary muscle movement. These cells, called "motor neurons," run from the brain through the brainstem or spinal cord to muscles that control movement in the arms, legs, chest, throat and mouth. In people with ALS, these cells die off, causing the muscle tissues to waste away. ALS does not affect a person's sensory functions or mental faculties. Other, nonmotor neurons, such as sensory neurons that bring information from sense organs to the brain, remain healthy.
Generally, ALS is categorized in one of two ways: Upper motor neuron disease affects nerves in the brain, while lower motor neuron disease affects nerves coming from the spinal cord or brainstem. In both cases, motor neurons are damaged and eventually die. ALS is fatal. The average life expectancy after diagnosis is two to five years, but some patients may live for years or even decades. (The famous physicist Stephen Hawking, for example, lived for more than 50 years after he was diagnosed.) There is no known cure to stop or reverse ALS.
Each person with ALS experiences a different proportion of upper and lower motor neurons that die. This results in symptoms that vary from person to person. The disease progresses, affecting more nerve cells as time goes on. As muscle tissues deteriorates, the muscles become weaker and atrophy (wither) and the person's limbs may begin to look thinner. However, the muscles can also become spastic (moving involuntarily) and this may lead to increased muscle tone in some parts of the body.
Although no one knows for sure, reports suggest 12,000 &ndash 15,000 people in the United States have ALS every year doctors tell about 5,000 people that they have it. Because records on ALS have not been kept throughout the country, it is hard to estimate the number of ALS cases in the United States. ALS is slightly more common in men than women. ALS is age related most people find out they have it when they are between 55 and 75 years of age, and live from 2 to 5 years after symptoms develop. How long a person lives with ALS seems to be related to age people who are younger when the illness starts live slightly longer. To read the latest prevalence of ALS for Oct. 2010 &ndash December 11, 2011, click here pdf icon .
About 5&ndash10% of ALS cases occur within families. This is called familial ALS and it means that two or more people in a family have ALS. Familial ALS is found equally among men and women. People with familial ALS usually do not fare as well as persons with ALS who are not related, and typically live only one to two years after symptoms appear.
This Chemical Is So Hot It Destroys Nerve Endings—in a Good Way
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In Morocco there grows a cactus-like plant that’s so hot, I have to insist that the next few sentences aren’t hyperbole. On the Scoville Scale of hotness, its active ingredient, resiniferatoxin, clocks in at 16 billion units. That’s 10,000 times hotter than the Carolina reaper, the world’s hottest pepper, and 45,000 times hotter than the hottest of habaneros, and 4.5 million times hotter than a piddling little jalapeno. Euphorbia resinifera, aka the resin spurge, is not to be eaten. Just to be safe, you probably shouldn’t even look at it.
But while that toxicity will lay up any mammal dumb enough to chew on the resin spurge, resiniferatoxin has also emerged as a promising painkiller. Inject RTX, as it’s known, into an aching joint, and it’ll actually destroy the nerve endings that signal pain. Which means medicine could soon get a new tool to help free us from the grasp of opioids.
The human body is loaded with different kinds of sensory neurons. Some flavors respond to light touch, others signal joint position, yet others respond only to stimuli like tissue injury and burns. RTX isn’t going to destroy the endings of all these neurons willy-nilly. Instead, it binds to a major molecule in specifically pain-sensing nerve endings, called TRPV1 (pronounced TRIP-vee one).
This TRPV1 receptor normally responds to temperature. But it also responds to a family of molecules called pungents, which includes capsaicin, the active ingredient in hot pepper. “So when you put hot pepper on your tongue and it feels like it's burning, it's not because your tongue is on fire,” says Tony Yaksh, a professor in the anesthesiology and pharmacology department at UC San Diego who’s studied RTX. “It's simply activating the same sensory axons that would have been activated if your tongue had been on fire.”
RTX is a capsaicin analog, only it’s between 500 and 1,000 times more potent. When RTX binds to TRPV1, it props open the nerve cell’s ion channel, letting a whole lot of calcium in. That’s toxic, leading to the inactivation of the pain-sensing nerve endings.
This leaves other varieties of sensory neurons unaffected, because RTX is highly specific to TRPV1. “So you gain selectivity because it only acts on TRPV1, which is only on a certain class of fibers, which only transmit pain,” says Yaksh. “Therefore you can selectively knock out pain without knocking out, say, light touch or your ability to walk.”
So if you wanted to treat knee pain, you could directly inject RTX into the knee tissue. You’d anesthetize the patient first, of course, since the resulting pain would be intense. But after a few hours, that pain wears off, and you end up with a knee that’s desensitized to pain.
Researchers have already done this with dogs. “It is profoundly effective there, and lasts much, much longer than I might have expected, maybe a median of 5 months before the owners of the dogs asked for reinjection,” says Michael Iadarola, who’s studying RTX at the National Institutes of Health. “The animals went from basically limping to running around.” One dog even went 18 months before its owners noticed the pain had returned.
That’s a very targeted application, but what about more widespread pain? Cancer patients, for instance, can live in agony through their end-of-life care. Here, too, RTX might work as a powerful painkiller. In fact, the NIH is in the midst of trials with bone cancer patients.
“We use the same technique for administering this as we would a spinal anesthetic,” says NIH anesthesiologist Andrew Mannes. “The whole idea is you're not injecting into the spinal column itself, you're injecting it into the fluid that surrounds the spinal column.” Injecting straight into the cord would damage it. Patients are anesthetized for all of this, and treated with short-term painkillers when they wake. “That seems to get them over the worst of it, and then over the next few hours it subsides until the point where they don't feel the pain any longer.”
Here RTX is working on the same principle as if you injected it straight into a specific area of concern like a knee. But because it’s injected more centrally, it delivers widespread pain relief. "For many of the cancer patients, we need to have the drug remove pain from a lot of different regions," says Iadarola. "So we give it into a compartment where the nerves to the lower half of the body are gathered together."
Now, the thing with pain is, it evolved for a reason. It’s an indispensable tool for you to feel if you’re doing something to your body that you shouldn’t be, like holding a scalding cup of coffee. Of course we want to alleviate pain, but might that be problematic if it’s too effective?
For people with knee pain, not really—the injection is targeting a specific area, so the rest of your body can still feel pain. And for end-of-life care, a central injection can bring long-awaited relief. “We're doing that on cancer pain patients who have tried all other treatments and they've not been successful,” says NIH neurosurgeon John Heiss. “The FDA has only allowed us to have the indication for cancer patients with limited life expectancy, because the concern is that if you lose pain and temperature sensation you could have deleterious effects.”
RTX’s promise lies in its specificity. Think of it like a sniper rifle for pain, whereas opioids are more like hand grenades. Opioids target receptors all over the body, not a specific kind of sensory neuron. “That's why when you give it to somebody, you get problems with constipation, sedation, they can have respiratory depression,” says Mannes.
That and you have to take opioids constantly, but not so with RTX. “You give it once and it should last for an extended period of time because it is destroying the fibers,” says Mannes. “But the other thing with this to remember is there's no reinforcement. There's no high associated with it, there's no addiction potential whatsoever.”
If RTX does become widely available, by the way, it’s not going to be for you to treat a sore post-marathon knee—this is a serious drug for serious conditions. But by more directly addressing the root of pain, a plant with a hell of a kick could help us cut back on opioids and other grenade-like painkillers.
Motor neurone disease
Motor neurone disease (MND) is a neurodegenerative disease that causes rapidly progressive muscle weakness. Specifically, the disease affects nerve cells (motor neurons) that control the muscles that enable you to move, speak, breathe and swallow.
MND is also known as Amyotrophic Lateral Sclerosis (ALS) or Lou Gehrig’s disease. Approximately 1400 people in Australia are living with this disease. MND typically affects people in their mid-50s and survival is approximately 2-5 years from the onset of symptoms. Although there is currently no cure for MND, an anti-glutamatergic medication is available and slows the progression of the disease.
We are trying to improve our understanding of what causes the neurons to die by studying patients with MND using novel electrical and magnetic tests. We also in the process of conducting a drug trial in the hope that it slows the progression of this devastating illness.
OUR LATEST RESEARCH
Frontotemporal dementia and motor neurone disease
This project’s objective is to develop cell culture and mouse models to study underlying pathomechanisms and develop/test new treatments.
Positional cloning of a chromosome 16 dementia / motor neurone disease gene
The aims of this project are to undertake the biological characterisation of this novel neurodegeneration gene. We are also examining a panel of commercially available and clinically relevant agonists and antagonists to modulate key pathways involved in Alzheimer’s disease and other neurodegenerative disorders.
The air, the water, the food and the surfaces around us are populated with microorganisms. Some are quite harmless, some are useful, and a percentage of them are quite dangerous. The latter group can cause serious illnesses that can damage our body, from simple colds to food poisonings. But thankfully, most people do not get sick easily. They have several defensive barriers in place. Besides those barriers, there is a complex defensive system that actively destroys potentially harmful organisms through several mechanisms. It is called the immune system.
Let us imagine there is a nasty microorganism outside that really wants to get inside our body. There are so many cells that can be invaded and used to its advantage, and our blood has all kinds of nutrients that a parasite would just die for. But in order to reach those treasures of ours, it has to get in first. And it is not that easy. Even before encountering the immune system, it has to overcome the passive barriers present.
First line of defense: skin, enzymes and acids
Look at yourself in the mirror. Our whole body is covered with a complex structure called the skin. It has multiple layers and is very hard to penetrate. You need a special instrument to break the skin – for example a proboscis of a mosquito, a claw, or a sting. Then the microorganisms can potentially invade.
Our face has several potential entry points for pathogens. The mouth and the eye can be typical examples. The eye is a very complex structure, and some of its most delicate components are constantly exposed to the outside – in order for us to see. And the mouth is, obviously, a place where the food enters. And with food, any kind of microorganisms can enter as well. But there is a solution! In areas where the body cannot form thick barriers – it uses chemistry. Our tears and our saliva have a special enzyme that can kill bacteria very effectively. It is called lysozyme. Our saliva also contains digestive enzymes that can both help digest the food and the bacteria it can potentially contain.
If you cannot destroy them – detain them. This approach is very effectively used in our noses and our ears. The mucous tissues in our noses produce a complex substance called mucus. It surrounds the dust and the potential pathogens and then we can eliminate them all when we sneeze. The cells in our ears use similar approach – they produce earwax. It also surrounds all foreign objects and then all of them can be easily eliminated.
Even if a microorganism has survived the tough environment of our mouths, it can potentially be destroyed later. When we digest food, the stomach is filled with extremely active acid. Most microorganisms cannot survive it.
But despite those effective mechanical and chemical barriers, the pathogens still get into our bodies. They have different ways to overcome our walls. And that is where the second line of defense is waiting for them – our immune system.
The Army Behind The Wall: The Immune System
Our immune system is unique. In the most systems of our body, we have a chain of organs, where each organ has its own specific function. The immune system is organized differently. It is spread around our body. And its main drivers are not organs, but immune cells. And these immune cells, in their turn, perform a variety of functions:
- destroy pathogens
- destroy infected, damaged and old cells
- destroy potential cancer cells
- produce antibodies
- produce antitoxins
As you can see, immune cells not only destroy any potential foreign organisms, they also support the integrity of the body – the homeostasis.
Our immune cells are also called white blood cells. There are several types of immune cells and all of them are produced in the bone marrow from special stem cells. These cells are also called hematopoetic, as they are the starting point of production of all cells in the blood – red blood cells and white blood cells.
Though there are multiple types of immune cells, they can be grouped into two major divisions depending on the way when and how they respond to the invasion: there are innate immunity cells and adaptive immunity cells.
Examples of innate and adaptive immunity cell
How are they different?
Innate immunity: First responders
The cells of innate immunity are the first on the spot. They are like patrol officers. They circulate in our bloodstream, are present in our lymph nodes and even in our lungs. There several major innate immunity cells:
- dendritic cells
Macrophages and neutrophils are phagocytic cells. After they identify the intruder, they surround it and use special enzymes and hydrogen peroxide to destroy it. This process is called phagocytosis.
The process of phagocytosis
Dendritic cells, in their turn, are not warriors. But they are very important as well. They are taking up pieces of the invading bacteria or viruses and push them up to their cellular membranes, so the receptors of other cells can interact with them. This process is called antigen presenting.
Anything can be an antigen – a foreign molecule, a molecule of the body itself, or even a whole pathogen such as a virus. And it is important to “present” it to other cells because of a very interesting mechanism our immune system has. You see, the innate immune cells are only interested if the object they meet is foreign or not. They only differentiate “self” from “non-self”. They are non – specific. But the cells of adaptive immunity know exactly who they are dealing with.
Adaptive immunity: special forces
The cells of adaptive immunity system are called lymphocytes. There are two main types of lymphocytes. Each type mounts their own specific defense against the invading enemy.
If a dendritic cell brings its piece of an antigen to a T-lymphocyte, or a T-cell, it would start a cellular immunity response. T-cells have specific receptors on their surfaces. Each particular T – cell has receptors for parts of a specific bacterium, virus or other agent. So when a dendritic cell shows its antigen, T-cells with suitable receptors flock to the area of infection. Then they destroy the bacteria or infected cells in question.
If our antigen presenting cell would go another type of lymphocyte called the B-lymphocyte or a B-cell, it would trigger humoral immunity response. B-cells do not kill directly. They produce specific proteins that can tightly stick to the pathogen – antibodies. Again, there are specific antibodies that react only with specific organisms. They are not universal. When the organism is “marked” with antibodies, it is much easier to kill and destroy.
What is great about adaptive immunity is the fact that if they ever encounter a particular agent of infection, they would remember it. And if it tries to invade again, they will mount the defense twice as rapidly and quickly clear it away. That is why we say that we have immunity to rubella or chickenpox – once we had this particular disease, the information gets written down by our immune cells. And next time, they would easily eliminate the same virus.
This same principle is behind vaccinations. A vaccine contains parts of certain viruses and bacteria. They are weak enough not to cause a real illness, but foreign enough to force our immune cells to produce antibodies to them. And when we actually encounter the infection, we either would not have it at all, or we would have only a mild illness, not a full-blown one with lots of complications.
Thankfully, our body is not that easy to destroy. But we also should remember that by supporting it in any way we can – eating nourishing meals, keeping clean and fit – we help it defend itself better.
References and further reading:
[4.] Janeway, C. A. et al. (2001) Immunobiology: The Immune System in Health and Disease. 5th edition. New York. Garland Science, 2001.
Ice Bucket Challenge funds gene discovery in ALS (MND) research
Scientists have identified a new gene contributing to the disease, NEK1.
The Ice Bucket Challenge has raised $115m (£87.7m) from people pouring cold water over themselves and posting the video on social media.
It was criticised as a stunt, but has funded six research projects.
Research by Project MinE, published in Nature Genetics, is the largest-ever study of inherited ALS, also known as motor neurone disease (MND).
More than 80 researchers in 11 countries searched for ALS risk genes in families affected by the disease.
"The sophisticated gene analysis that led to this finding was only possible because of the large number of ALS samples available," Lucie Bruijn of the ALS Association says.
The identification of gene NEK1 means scientists can now develop a gene therapy treating it.
Although only 10% of ALS patients have the inherited form, researchers believe that genetics contribute to a much larger percentage of cases.
Social media was awash with videos of people pouring cold water over their heads to raise money for ALS in the summer of 2014.
More than 17 million people uploaded videos to Facebook, including many celebrities who rose to the challenge, which were then watched by 440 million people worldwide.
How silver ions kill bacteria
The antimicrobial properties of silver have been known for centuries. While it is still a mystery as to exactly how silver kills bacteria, University of Arkansas researchers have taken a step toward better understanding the process by looking at dynamics of proteins in live bacteria at the molecular level.
Traditionally, the antimicrobial effects of silver have been measured through bioassays, which compare the effect of a substance on a test organism against a standard, untreated preparation. While these methods are effective, they typically produce only snapshots in time, said Yong Wang, assistant professor of physics and an author of the study, published in the journal Applied and Environmental Microbiology.
Instead, Wang and his colleagues used an advanced imaging technique, called single-particle-tracking photoactivated localization microscopy, to watch and track a particular protein found in E. coli bacteria over time. Researchers were surprised to find that silver ions actually sped up the dynamics of the protein, opposite of what they thought would happen. "It is known that silver ions can suppress and kill bacteria we thus expected that everything slowed down in the bacteria when treated with silver. But, surprisingly, we found that the dynamics of this protein became faster."
The researchers observed that silver ions were causing paired strands of DNA in the bacteria to separate, and the binding between the protein and the DNA to weaken. "Then the faster dynamics of the proteins caused by silver can be understood," said Wang. "When the protein is bound to the DNA, it moves slowly together with the DNA, which is a huge molecule in the bacteria. In contrast, when treated with silver, the proteins fall off from the DNA, moving by themselves and thus faster."
The observation of DNA separation caused by silver ions came from earlier work that Wang and colleagues had done with bent DNA. Their approach, now patent pending, was to put strain on DNA strands by bending them, thus making them more susceptible to interactions with other chemicals, including silver ions.
The National Science Foundation-funded study validated the idea of investigating the dynamics of single proteins in live bacteria, said Wang, an approach that could help researchers understand the real-time responses of bacteria to silver nanoparticles, which have been proposed for fighting against so-called "superbugs" that are resistant to commonly prescribed antibiotics.
"What we want to do eventually is to use the new knowledge generated from this project to make better antibiotics based on silver nanoparticles," said Wang.
List of 9 Important Viral Diseases | Diseases | Human Health | Biology
List of nine important viral diseases seen in humans: 1. Chickenpox 2. Smallpox 3. Poliomyelitis 4. Measles 5. Mumps 6. Rabies 7. Trachoma 8. Influenza 9. Hepatitis.
Viral Disease # 1. Chickenpox (Varicella):
Pathogen – Herpes-zoster virus (DNA- virus)
Epidemiology – Contagious & Formite borne
Incubation Period – 12-20 days
Symptoms – Dark red coloured rash or pox changing into vesicles, crusts and falling.
Prophylaxis – Now vaccine available, isolation.
Therapy – Zoster Immunoglobulins (ZIG).
Viral Disease # 2. Smallpox (Variolla):
Pathogen – Variola-virus (DNA-Virus)
Epidemiology – Contagious & Droplet infection
Incubation Period – 12-days
Symptoms – Appearance of rash changing into pustules, scabs and falling.
Prophylaxis – Smallpox vaccine.
Therapy – No case reported after 1978.
Viral Disease # 3. Poliomyelitis:
Pathogen – Polio-virus (RNA-virus)
Epidemiology – Direct & oral
Incubation Period – 7-14 days
Symptoms – Damages motor neurons causing stiffness of neck, convulsion, paralysis of generally legs.
Prophylaxis – ‘Salk’ vaccine and Oral Polio vaccine.
Viral Disease # 4. Measles (Rubeolla-Disease):
Pathogen – Rubeolla-virus (RNA-virus)
Epidemiology – Contagious & Droplet infection
Incubation Period – 10 days
Symptoms – Rubeolla (skin eruptions), coughing, sneezing etc.
Prophylaxis – Edmonston- B-vaccine, isolation
Therapy – Antibiotics & sulpha drugs.
Viral Disease # 5. Mumps:
Pathogen – Mumps-virus (RNA-virus)
Epidemiology – Contagious & Droplet infection
Incubation Period – 12-26 days
Symptoms – Painful enlargement of parotid salivary glands.
Prophylaxis – Mumps- vaccine isolation
Viral Disease # 6. Rabies (Hydrophobia):
Pathogen – Rabies-virus (RNA-virus)
Epidemiology – Indirect & inoculative (vectors are rabid animals monkeys, cats, dogs)
Incubation Period – 10 days to 1- 3 months
Symptoms – Spasm of throat & chest muscles, fears from water, paralysis and death.
Prophylaxis – Immunization of dogs.
Therapy – Pasteur- treatment (14 vaccines in stomach).
Viral Disease # 7. Trachoma:
Pathogen – Chlamydia trachomatis
Epidemiology – Contagious, formite-borne and flies (vectors)
Incubation Period – 5-12 days
Symptoms – Inflammation of conjunctiva & cornea leading to blindness.
Therapy – Tetracycline & sulfonamide.
Viral Disease # 8. Influenza (Flu):
Pathogen – Myxovirus influenzae (RNA-virus)
Epidemiology – Air borne and pandemic
Incubation Period – 24-48 Hours Lasts for 4-5 days
Symptoms – Bronchitis, sneezing bronchopneumonia, leucopenia, coughing, etc.
Therapy – Antibiotic therapy.
Viral Disease # 9. Hepatitis (Epidemic Jaundice):
Pathogen – Hepatitis-B virus
Epidemiology – Direct & oral (with food and water)
Incubation Period – 20-35 days
Symptoms – Damage to liver cells releasing bilirubin which causes jaundice.
Prophylaxis – Proper sanitation proper coverage of food, water, milk etc. use of chlorinated or boiled water, etc.
Active Vs Passive Immunity
The difference between active and passive immunity is simply where the antibodies came from. In active immunity, the immune cells of the body recognize foreign particles and cells and create antibodies to combat them. Passive immunity, on the other hand, simply gives an organism the correct antibodies to combat germs and pathogens. Passive immunity is most commonly seen in pregnancy when a mother’s antibodies pass to the baby and protect it. The baby’s active immunity is not developed yet, so it needs its mother’s antibodies.
Types of Recombinant Vaccines: 3 Types
Recombinant DNA technology in recent years has become a boon to produce new generation vaccines. By this approach, some of the limitations (listed above) of traditional vaccine production could be overcome. In addition, several new strategies, involving gene manipulation are being tried to create novel recombinant vaccines.
Types of Recombinant Vaccines:
The recombinant vaccines may be broadly categorized into three groups:
1. Subunit recombinant vaccines:
These are the components of the pathogenic organisms. Subunit vaccines include proteins, peptides and DNA.
2. Attenuated recombinant vaccines:
These are the genetically modified pathogenic organisms (bacteria or viruses) that are made non-pathogenic and used as vaccines.
3. Vector recombinant vaccines:
These are the genetically modified viral vectors that can be used as vaccines against certain pathogens. Some of the developments made in the production of recombinant vaccines against certain diseases are briefly described.
Type # 1. Subunit Vaccines:
As already stated, subunit recombinant vaccines are the components (proteins, peptides, DNAs) of the pathogenic organisms. The advantages of these vaccines include their purity in preparation, stability and safe use. The disadvantages are — high cost factor and possible alteration in native conformation. Scientists carefully evaluate the pros and cons of subunit vaccines for each disease, and proceed on the considered merits.
Hepatitis B is a widespread disease in man. It primarily affects liver causing chronic hepatitis, cirrhosis and liver cancer. Hepatitis B virus is a 42 nm particle, called Dane particle. It consists of a core containing a viral genome (DNA) surrounded by a phospholipid envelope carrying surface antigens (Fig. 16.1 A).
Infection with hepatitis B virus produced Dane particles and 22 nm sized particles. The latter contain surface antigens which are more immunogenic. It is however, very difficult to grow hepatitis B virus in mammalian cell culture and produce surface antigens. The gene encoding for hepatitis B surface antigen (HBsAg) has been identified. Recombinant hepatitis B vaccine as a subunit vaccine, is produced by cloning HbsAg gene in yeast cells. Saccharomyces cerevisiae, a harmless baking and brewing yeast, is used for this purpose (Fig. 16.1B). The gene for HBsAg is inserted (pMA 56) which is linked to the alcohol dehydrogenase promoter. These plasmids are then transferred and cultured.
The cells grown in tryptophan, free medium are selected and cloned. The yeast cells are cultured. The HBsAg gene is expressed to produce 2nm sized particles similar to those found in patients infected with hepatitis B. (These particles are immunoreactive with anti-HBsAg antibodies). The subunit HBsAg as 22 nm particles can be isolated and used to immunize individuals against hepatitis B.
Hepatitis B vaccine-the first synthetic vaccine:
In 1987, the recombinant vaccine for hepatitis B (i.e. HBsAg) became the first synthetic vaccine for public use. It was marketed by trade names Recombivax and Engerix-B. Hepatitis B vaccine is safe to use, very effective and produces no allergic reactions. For these reasons, this recombinant vaccine has been in use since 1987.
The individuals must be administered three doses over a period of six months. Immunization against hepatitis B is strongly recommended to anyone coming in contact with blood or body secretions. All the health professionals—physicians, surgeons, medical laboratory technicians, nurses, dentists, besides police officers, firefighters etc., must get vaccinated against hepatitis B.
Hepatitis B vaccine in India:
India is the fourth country (after USA, France and Belgium) in the world to develop an indigenous hepatitis B vaccine. It was launched in 1997, and is now being used.
Hepatitis B vaccine tomato?
Biotechnologists have been successful in inserting hepatitis B gene into the cells of the tomato plant. These genetically engineered plants produce hepatitis B antigens. The day may not be far off to get immunized against hepatitis B by having a tomato with lunch!
Foot and Mouth Disease:
Foot and mouth disease (FMD) is a highly contagious disease affecting cattle and pigs. A formalin killed foot and mouth disease virus (FMDV) was previously used to vaccinate against this disease. The genome of FMDV is composed of a single— stranded RNA, covered by four viral proteins (VP1, VP2, VP3 and VP4). Among these, VPI is immunogenic. The nucleotide sequence encoding VPI was identified in the FMDV genome. A double- stranded complementary DNA (cDNA) from the single-stranded viral RNA (genome) was synthesized.
This cDNA was then digested with restriction enzymes and the fragments were cloned by using plasmid pBR322 in E. coli. The recombinant vaccine for FMDV in the form of viral protein 1 was used to vaccinate animals. However, VPI vaccination was found to be less effective than that of the whole virus in protecting FMD. Further, studies are being pursued to improve the efficiency of subunit vaccine.
The concept of peptide vaccines:
Theoretically, it is expected that only small portions of a given protein (i.e., domains) are immunogenic and bind to antibodies. Logically, it is possible to use short peptides that are immunogenic as vaccines. These are referred to as peptide vaccines.
Peptide vaccines for foot and mouth disease:
Some details on the FMD are described above. The domains of viral protein I (VPI) of FMDV were chemically synthesized. From the C-terminal end of VPI, amino acids 141 to 160, 151 to 160 and 200 to 213, and from N-terminal end, amino acids 9 to 24, 17 to 32 and 25 to 41 were synthesized.
Each one of these short peptides (domains) was bound to the surface of a carrier protein (Fig. 16.2) and used as a vaccine. Among the peptides used, the one corresponding to amino acids 141 to 160 was found to be effective in immunizing guinea pigs against FMD. In addition, when two peptides were joined together (amino acids 141 to 158, and amino acids 200 to 213), they served as more efficient recombinant vaccines.
The success so far to use recombinant peptides as vaccines has been very limited. This is mainly because a short peptide usually is not enough to be sufficiently immunogenic, since it may not have the same conformation as that of the original viral particle. However, scientists continue their search for specific, inexpensive and safe synthetic peptide vaccines for various diseases.
Herpes Simplex Virus:
Herpes simplex virus (HSV) is an oncogenic (cancer-causing) virus. In addition, it also causes sexually transmitted diseases, encephalitis and severe eye infections. Attempts have been made to produce subunit vaccines against HSV. An envelope glycoprotein D (gD) of HSV that can elicit antibody production has been identified. This is a membrane bound protein, and difficult to isolate and purify. The glycoprotein D was modified by deleting the trans membrane portion of the protein (Fig. 16.3) and the gene was modified.
This gene for gD was cloned in a mammalian vector and expressed in Chinese hamster ovary (CHO). The advantage here is that the protein can get glycosylated (unlike in E. coli system). In the experimental trials, the modified form of gD was found to be effective against HSV.
Tuberculosis is caused by the bacterium Mycobacterium tuberculosis. It is often fatal, and as per some estimates nearly 3 million deaths occur every year due to this highly infectious disease. Antibiotics are used to treat tuberculosis. However, drug-resistant M. tuberculosis strains have been developed making the drug therapy sometimes ineffective. Vaccination for tuberculosis is therefore, advocated.
Bacillus Calmette-Guerin (BCG) vaccine:
In some countries, particularly the developing ones, BCG vaccine is widely used to protect against tuberculosis. However, countries like United States have not approved BCG vaccination for various reasons. BCG vaccine itself causes tuberculosis in some individuals (AIDS victims) and the vaccinated people respond positively for laboratory diagnosis of tuberculosis.
The secretory (extracellular proteins of M. tuberculosis have been purified and used for immuno-protection against tuberculosis. Of about 100 such proteins, six were found to be useful (either individually or in combination) to immunize guinea pigs. Attempts are underway to develop recombinant subunit vaccine against tuberculosis.
Group B strain of meningococci, namely Neisseria meningitidis causes meningitis in adolescents and young adults. Meningitis is characterized by inflammation of the membranes covering brain and spinal cord. The symptoms include headache, photophobia, irritability, and neck stiffness.
Pizza et al (2000) made a novel approach to develop a vaccine against meningitis. They identified 350 proteins (potential protective antigens) and the entire sequence of genome coding for these proteins in N. meningitidis. All the 350 candidate antigens were expressed in E. coli, purified and used to immunize mice. A good bactericidal antibody response was observed in these mice.
AIDS (Acquired Immunodeficiency Syndrome):
AIDS is a retroviral disease caused by human immunodeficiency virus (HIV). This disease is characterized by immunosuppression, neo-plasma and neurological manifestations. AIDS is invariably fatal, since as of now there is no cure. Development of a vaccine against AIDS is a top priority by DNA technologists world over. In fact, vaccines are being continuously developed and field tested, although there has been no success so far.
The development of two subunit vaccines, specifically the glycoproteins of HIV envelope is described here. The functions of gp120 and gp41 of HIV are illustrated in Fig. 16.4A. The glycoprotein gp120 projects out of the HIV envelope while the other glycoprotein gp41 lies beneath gp120.
On entering the body, the HIV binds to the host cells (T-lymphocytes) by attaching gp120 to the CD4 receptor sites on the cell surface. This attachment uncovers gp41 molecules and the viral envelope. Now gp41 binds to the host cell surface and opens a passage for the entry of the virus into the cell.
Biotechnologists have isolated the genes for gp120 and gp41 and inserted them into the bacterium E. coli. These bacterial cells produce gp120 and gp41 that can be used as recombinant vaccines against AIDS. The action of gp120 and gp41 in immunizing host T-lymphocytes is depicted in Fig. 16.4B.
The gp120 molecules stimulate the host immune system to produce anti- gp120 antibodies. These antibodies bind to gp120 and prevent its attachment to CD4. In a comparable manner, gp41 molecules also result in the production of anti-gp41 antibodies. These antibodies also bind to gp41 and block the virus- host cell union. The net result of using gp120 and gp41 vaccines is that the entry of HIV into the host cells is prevented.
Vaccine against AIDS— not yet a reality:
The description of vaccine development against AIDS (given above), which appears attractive is not so simple. The most important limitations are that the HIV has high frequency of mutations. Therefore the vaccines developed cannot bind to the new virus (i.e., mutated one). In addition, gp120 and gp41 are very poor stimulators of immune system. Despite these limitations, scientists have not lost hope, and continue their research to develop vaccines against AIDS.
DNA Vaccines (Genetic Immunization):
Genetic immunization by using DNA vaccines is a novel approach that came into being in 1990. The immune response of the body is stimulated by a DNA molecule. A DNA vaccine consists of a gene encoding an antigenic protein, inserted onto a plasmid, and then incorporated into the cells in a target animal.
The plasmid carrying DNA vaccine normally contains a promoter site, cloning site for the DNA vaccine gene, origin of replication, a selectable marker sequence (e.g. a gene for ampicillin resistance) and a terminator sequence (a poly—A tail).
DNA vaccine—plasmids can be administered to the animals by one of the following delivery methods.
ii. Intramuscular injection
iii. Intravenous injection
v. Gene gun or biolistic delivery (involves pressure delivery of DNA-coated gold beads).
DNA Vaccine and Immunity:
An illustration of a DNA vaccine and the mechanism of its action in developing immunity is given in Fig. 16.5. The plasmid vaccine carrying the DNA (gene) for antigenic protein enters the nucleus of the inoculated target cell of the host. This DNA produces RNA, and in turn the specific antigenic protein. The antigen can act directly for developing humoral immunity or as fragments in association with major histocompatibility class (MHC) molecules for developing cellular immunity.
As the antigen molecules bind to B- lymphocytes, they trigger the production of antibodies which can destroy the pathogens. Some of the B-lymphocytes become memory cells that can protect the host against future infections.
The protein fragments of the antigen bound to MHC molecules can activate the cytotoxic T-lymphocytes. They are capable of destroying the infected pathogenic cells. Some of the activated T-lymphocytes become memory cells which can kill the future infecting pathogens.
Complementary DNA vaccines:
For genetic immunization, complementary DNA (cDNA) vaccines can also be used. Some workers have successfully used cDNA as vaccines e.g. immunization of mice against influenza.
DNA vaccines for production of antigens and antibodies:
A novel approach for the production of antigens as well as antibodies by DNA vaccine was developed in 1997. In the experiments conducted in mice, researchers injected plasmids containing the genes for malarial parasite and also the genes for the antibodies against malarial parasite. The B-lymphocytes 4 of these mice performed a double duty, and produced antigens for and antibodies against malarial parasite.
The antigens stimulate to produce more and more antibodies. The antibodies so produced react with malarial parasite. The generation of antigens and antibodies by using a DNA vaccine is a recent development in immunology, and is referred to as antigenic antibody approach of DNA vaccine.
Screening of pathogenic genome for selecting DNA vaccines:
The ultimate goal of scientists is to choose the right DNA fragment from the pathogen to serve as a vaccine for the strongest immune response against the invading pathogen. For this purpose, the pathogen’s DNA can be broken into fragments and a large number of vaccines DNA—plasmids can be prepared.
The immune response for each one of the DNA vaccines can be studied by injecting the pathogen. By screening the DNA fragments of the pathogenic genome, it is possible to choose one or few DNA vaccines that can offer maximal immune protection.
Advantages of DNA vaccines:
There are several advantages of using DNA vaccines in immunization:
1. The tedious and costly procedures of purifying antigens or creating recombinant vaccines are not necessary.
2. DNA vaccines are very specific in producing the target proteins (antigens or antibodies). Thus, they trigger immune response only against the specific pathogen.
3. In general, DNA vaccines elicit much higher immune response compared to other kinds of vaccines.
4. DNA vaccines are more stable for temperature variations (low or high) than the conventional vaccines. Thus, the storage and transport problems associated with vaccines are minimal.
5. The delivery methods to the host are simpler for DNA vaccines.
Disadvantages of DNA vaccines:
1. The fate of the DNA vaccine in the host cells is not yet clear. There is a possibility of this DNA getting integrated into the host genome and this may interrupt the normal functions.
2. There also exists a danger of cancer due to DNA vaccines.
3. The post-translational modification of the gene (DNA vaccine) product in host cells may not be the same as that found in the native antigen.
Present status of DNA vaccines:
Since 1990, several groups of workers world-over have been trying to develop DNA vaccines against various diseases in experimental animals. Genetic immunization has been done against a number of pathogenic organisms. These include influenza A virus, rabies virus, hepatitis B virus, bovine herpes virus, HIV type I, and Plasmodium species (malarial parasite). It must be noted that DNA vaccines have not been tried in humans for obvious reasons. The most important being the unknown risks of these foreign DNAs in human subjects.
Several workers are trying to use RNA molecules as vaccines. These RNAs can readily synthesize the antigenic proteins and offer immunity. But unfortunately, RNAs are less stable than DNAs. This poses a big problem for RNA vaccine manufacture and distribution. Therefore, the progress in the development of RNA vaccines has been rather slow compared to DNA vaccines.
Plants as Edible Subunit Vaccines:
Plants serve as a cheap and safe production systems for subunit vaccines. The edible vaccines can be easily ingested by eating plants. This eliminates the processing and purification procedures that are otherwise needed. Transgenic plants (tomato, potato) have been developed for expressing antigens derived from animal viruses (rabies virus, herpes virus). A selected list of recombinant vaccines against animal viruses produced in plants is given in Table 16.2.
Edible vaccine production and use:
The production of vaccine potatoes is illustrated in Fig. 16.6. The bacterium, Agrobacterium tumefaciens is commonly used to deliver the DNA (genetic material) for bacterial or viral antigens. A plasmid carrying the antigen gene and an antibiotic resistance gene are incorporated into the bacterial cells (A. tumefaciens).
The cut pieces of potato leaves are exposed to an antibiotic which can kill the cells that lack the new genes. The surviving cells (i.e., gene altered ones) can multiply and form a callus (clump of cells). This callus is allowed to sprout shoots and roots, which are grown in soil to form plants.
In about three weeks, the plants bear potatoes with antigen vaccines. The first clinical trials in humans, using a plant- derived vaccine were conducted in 1997. This involved the ingestion of transgenic potatoes with a toxin of E. coli causing diarrhea.
Type # 2. Attenuated Recombinant Vaccines:
In the early years of vaccine research, attenuated strains of some pathogenic organisms were prepared by prolonged cultivation — weeks, months or even years. Although the reasons are not known, the infectious organism would lose its ability to cause disease but retains its capability to act as an immunizing agent. This type of approach is almost outdated now.
It is now possible to genetically engineer the organisms (bacteria or viruses) and use them as live vaccines, and such vaccines are referred to as attenuated recombinant vaccines. The genetic manipulations for the production of these vaccines are broadly of two types:
1. Deletion or modification of virulence genes of pathogenic organisms.
2. Genetic manipulation of non-pathogenic organisms to carry and express antigen determinants from pathogenic organisms.
The advantage with attenuated vaccines is that the native conformation of the immunogenic determinants is preserved hence the immune response is substantially high. This is in contrast to purified antigens which often elicit poor immunological response.
Some of the important attenuated vaccines developed by genetic manipulations are briefly described.
Cholera is an intestinal disease characterized by diarrhea, dehydration, abdominal pain and fever. It is caused by the bacterium, Vibrio cholera. This pathogenic organism is transmitted by drinking water contaminated with fecal matter. Cholera epidemics are frequently seen in developing countries where the water purification and sewage disposal systems are not well developed.
On entering the small intestine, V. cholera colonizes and starts producing large amounts of a toxic protein, a hexameric enterotoxin. This enterotoxin stimulates the cells lining intestinal walls to release sodium, bicarbonate and other ions. Water accompanies these ions leading to severe diarrhea, dehydration, and even death.
The currently used cholera vaccine is composed of phenol-killed V. cholera. The immuno-protection, lasting for 3-6 months is just moderate. Attempts are being made to develop better vaccines. The DNA technologists have identified the gene encoding enterotoxin (toxic protein). Enterotoxin, an hexamer, consists of one A subunit and five identical B subunits. The A subunit has two functional domains-the A1 peptide which possesses the toxic activity and A2 peptide that joins A subunit to B subunits.
By genetic engineering, it was possible to delete the DNA sequence encoding A1 peptide and create a new strain of V. cholera. This strain is non-pathogenic, since it cannot produce enterotoxin. The genetically engineered V. cholera is a good candidate to serve as an attenuated vaccine.
Creating a new strain of V. cholera:
The development of a new strain of Vibrio cholera that can effectively serve as an attenuated recombinant vaccine is depicted in Fig. 16.7, and briefly described below.
1. A tetracycline resistance gene was inserted into the A1 peptide sequence of V. cholera chromosome. This destroys the DNA sequence encoding for A1 peptide, besides making the strain resistant to tetracycline. Unfortunately, the tetracycline resistant gene is easily lost and the enterotoxin activity is restored. Because of this, the new strain of V. cholera as such cannot be used as a vaccine.
2. The DNA sequence of A1 peptide is incorporated into a plasmid, cloned and digested with restriction enzymes (Clal and Xbal). In this manner, the A1 peptide coding sequence is deleted (the DNA encoding for 183 of the 194 amino acids of the A1 peptide is actually removed). By using T4 DNA ligase, the plasmid is re-circularized. This plasmid contains a small portion of A1 peptide coding sequence.
3. The plasmid, containing the deleted A1 peptide sequence is transferred by conjugation into the V. cholera strain carrying a tetracycline resistance gene.
4. Recombination can occur between the plasmid (containing a small portion of peptide A1 coding sequence) and the chromosome of V. cholera (carrying tetracycline resistance gene). The result of this double crossover is the formation of V. cholera containing a chromosomal DNA lacking A1 peptide DNA sequence. As the bacterium, V. cholera multiplies, the plasmids are lost in the next few generations.
5. The V. cholera cells defective in A1 peptide are selected, based on tetracycline sensitivity. It may be noted that this new strain lacks tetracycline resistance gene.
The genetically engineered V. cholera cells with deleted A1 peptide DNA sequence are quite stable. They cannot produce active enterotoxin but possess all other biochemical functions of the pathogen. This new strain of V. cholera is undergoing trials for its efficiency as a vaccine. Preliminary results indicate that this attenuated vaccine can protect about 90% of the volunteers against cholera. But there are some side effects. Scientists continue their work to develop a better vaccine against cholera.
Potato as a vehicle for cholera vaccine:
A group workers have developed a gene altered potato containing attenuated cholera vaccine. These potatoes when fed to mice induced immunity against cholera.
The different strains of Salmonella genus are responsible for causing typhoid, enteric fever, food poisoning and infant death. Immunoprotection against Salmonella pathogens is really required. Some workers have been successful in deleting aro genes and pur genes in Salmonella.
Aro genes encode for the enzymes responsible for the biosynthesis of aromatic compounds, while pur genes encode for enzymes of purine metabolism. The new strains of Salmonella can be grown in vitro on a complete medium.
The doubly deleted strains have a very restricted growth in vivo, while they can stimulate immunological response. The genetically engineered attenuated vaccines of Salmonella have been shown to be effective as oral vaccines in experimental animals (mice, cattle, sheep, and chickens). Some workers claim that the new strain of Salmonella offers immunoprotection in humans also.
Leishmania species are flagellated protozoan parasites and are responsible for the disease leishmaniasis. This disease is characterized by cutaneous, visceral and mucosal leisons. Leishmaniasis is transmitted by sand flies.
An attenuated strain of leishmania has been created and successfully used in mice to offer immunoprotection against leishmaniasis. In Leishmania major, the genes encoding dihydrofolate reductase-thymidylate synthase can be replaced by the genes encoding resistance to antibiotics G-418 and hygromycin.
This new strain of L. major invariably requires thymidine in the medium for its growth and multiplication. The attenuated strain of L. major can survive only a few days when administered to mice. This short period is enough to induce immunity in mice against the lesions of leishmania. However, more experiments on animals have to be carried out before the leishmania attenuated vaccine goes for human trials.
Type # 3. Vector Recombinant Vaccines:
Some of vectors can be genetically modified and employed as vaccines against pathogens.
Vaccines against Viruses- Vaccinia Virus:
Vaccinia viruses is basically the vaccine that was originally used by Jenner for the eradication of smallpox. The molecular biology of this virus has been clearly worked out. Vaccinia virus contains a double-stranded DNA (187 kb) that encodes about 200 different proteins. The genome of this virus can accommodate stretches of foreign DNA which can be expressed along with the viral genes.
The vaccinia virus can replicate in the host cell cytoplasm (of the infected cells) rather than the nucleus. This is possible since the vaccinia virus possesses the machinery for DNA replication, transcription-DNA polymerase, RNA polymerase etc. The foreign genes inserted into the vaccinia virus can also be expressed along with the viral genome. Thus, the foreign DNA is under the control of the virus, and is expressed independently from the host cell genome.
The vaccinia viruses are generally harmless, relatively easy to cultivate and stable for years after lyophilization (freeze-drying). All these features make the vaccinia virus strong candidates for vector vaccine. The cloned foreign genes (from a pathogenic organism) can be inserted into vaccinia virus genome for encoding antigens which in turn produces antibodies against the specific disease- causing agent.
The advantage with vector vaccine is that it stimulates B-lymphocytes (to produce antibodies) and T-lymphocytes (to kill virus infected cells). This is in contrast to a subunit vaccine which can stimulate only B-lymphocytes. Thus, vaccinia virus can provide a high level of immunoprotection against pathogenic organisms. Another advantage of vaccinia virus is the possibility of vaccinating individuals against different diseases simultaneously. This can be done by a recombinant vaccinia viruses which carries genes encoding different antigens.
Antigen genes for certain diseases have been successfully incorporated into vaccinia virus genome and expressed. Thus, vector vaccines have been developed against hepatitis, influenza, herpes simplex virus, rabies, angular stomatitis virus and malaria. However, none of these vaccines has been licensed for human use due to fear of safety. It is argued that recombinant vaccinia virus might create life threatening complications in humans.
Production of recombinant vaccinia viruses:
The development of recombinant vaccinia virus is carried out by a two-step procedure (Fig. 16.8).
1. Assembly of plasmid insertion vector:
Fresh vaccinia (cow pox) viruses are processed to release their DNAs. Now genes from hepatitis B virus, herpes simplex virus and influenza virus are added one after another and inserted into vaccinia virus genome. These DNA clusters are cloned in E. coli for increasing their number and to produce plasmid insertion vectors. The plasmid contains the foreign subunit genes, the natural vaccinia genes, including the promoter genes. The recombinant plasmids are isolated and purified and serve as plasmid insertion vectors.
2. Production of recombinant vaccinia viruses:
The animal cells are infected with plasmid insertion vectors and normal vaccinia viruses. As the viral replication occurs, the plasmids are taken up to produce recombinant vaccinia viruses. The plasmid insertion vector incorporates its genes into vaccinia virus genome at a place that encodes for the enzyme thymidine kinase (TK).
Thus the recombinant viruses have lost their ability to produce TK. There are two advantages of loss of TK gene. One is that it is easy to select recombined vaccinia viruses that lack TK gene and the second is that these viruses are less infectious than the normal viruses. The recombinant vaccinia viruses, released from the cultured animal cells, can be successfully used as vaccines. These live viral vaccines have some advantages over the killed or subunit vaccines.
1. Authenticated antigens that closely resemble natural antigens can be produced.
2. The virus can replicate in the host cells. This enables the amplification of the antigens for their action on B-lymphocytes and T-lymphocytes.
3. There is a possibility of vaccinating several diseases with one recombinant vaccinia virus.
1. The most important limitation is the yet unknown risks of using these vaccines in humans.
2. There may be serious complications of using recombinant viral vaccines in immunosuppressed individuals such as AIDS patients.
Other viral recombinant vaccines:
Most of the work on the development of live viral vaccines has been carried out on vaccinia virus. Other viruses such as adenovirus, poliovirus and varicella-zoster virus are also being tried as recombinant vaccines. Scientists are attracted to develop a recombinant poliovirus as it can be orally administered. It might take many more years for the recombinant viral vaccines to become a reality for human use.
Delivery of Antigens by Bacteria:
It is known that the antigens located on the surface of a bacterial cell are more immunogenic than the antigens in the cytoplasm. Based on this observation, scientists have developed strategies to coat the surfaces of non-pathogenic organisms with antigens of pathogenic bacteria.
Flagellin is a protein present in the fragella (thread like filaments) of Salmonella. A synthetic oligonucleotide encoding the epitope of cholera toxin B subunit was inserted into Salmonella flagellin gene. This epitope was in fact found on the flagellum surface. These flagella-engineered bacteria, when administered to mice, raised antibodies against the cholera toxin B subunit peptide. It may be possible in future to incorporate multiple epitopes (2 or 3) into the flagellin gene to create multivalent bacterial vaccines.