Kissing the Epstein-Barr virus goodbye?

Key text

But we might not have to give up kissing just yet. A vaccine currently under development in Australian laboratories could help limit the dangers posed by this sometimes deadly virus.

How was the virus discovered?

In 1961, a surgeon working in Uganda, Denis Burkitt, presented the results of his research to staff at the Middlesex Hospital Medical School in Britain. He reported that the incidence of a certain tumour in African children had a geographic distribution corresponding to rainfall and temperature patterns.

The disease, which affects about 8 in every 100,000 children in parts of Africa and Papua New Guinea, quickly became known as Burkitt’s lymphoma. The influence of climate on its incidence seemed to suggest that some biological factor was involved. Three researchers, M.A. Epstein, Y.M. Barr and B.G. Achong, immediately began looking for possible cancer-causing viruses in samples of the tumour sent from Uganda to Britain.

In 1964, they identified the culprit using an electron microscope: a previously unknown member of the herpes family of viruses. Epstein and Barr were awarded the dubious honour of having the pathogen named after them.

What diseases does the virus cause?

The Epstein-Barr virus is thought to be responsible for a number of diseases in addition to glandular fever (otherwise known as infectious mononucleosis) and Burkitt’s lymphoma. One of these is nasopharyngeal carcinoma: this is a tumour of the nasal passages and throat which affects up to 2 per cent of people in southern China and also occurs in Southeast Asia, northern Africa and among Arctic peoples. It has been proposed as a possible cause of Hodgkin’s disease (a type of cancer affecting cells of lymph nodes).

Diseases caused by the virus are particularly common among people with reduced immunity. For example, the virus is associated with ‘post-transplant lymphoproliferative disease’, a tumour often found in organ transplant patients. The immune systems of such patients are usually suppressed artificially by drug therapy to help prevent the body from rejecting the new organ.

AIDS sufferers, who also have reduced immunity, commonly suffer from ‘oral hairy leukoplakia’, a condition involving considerable replication of the Epstein-Barr virus in cells along the edge of the tongue. And researchers have suggested that the high incidence of malaria in countries where Burkitt’s lymphoma is prevalent may also play a role in the disease by suppressing the body’s immune system.

Scientists don’t know why the virus causes a relatively mild disease like glandular fever in some people and malignant tumours in others. Some evidence suggests that genetic factors may play a role.

How is the virus spread?

People infected with the Epstein-Barr virus will retain it for life, but it may not make them sick. In fact, the virus infects almost everyone in developing countries and more than 80 per cent of people in developed countries. It is spread mainly via the transfer of saliva between individuals, which is the reason that glandular fever has been dubbed the ‘kissing disease’.

Most people are infected with the virus during childhood, probably by their mothers, and are usually not noticeably affected. On the other hand, people infected for the first time during or after adolescence (10–20 per cent of people living in developed countries) have a 50 per cent chance of contracting glandular fever.

How does the virus work?

The Epstein-Barr virus appears capable of infecting only two major cell types: the outer (epithelial) cells of the salivary gland, and white blood cells known as B lymphocytes (B-cells). Infection with the Epstein-Barr virus develops first in the salivary gland. Large amounts of the virus are released in the saliva, enabling it to spread from one person to another.

Infection of B-cells with the virus causes them to proliferate. This proliferation is controlled by the immune system; if the correct immune response does not develop, individuals are at risk of developing a form of cancer.

The response of the body’s immune system

The Epstein-Barr virus produces about 100 different antigens (large protein molecules) during the active phase of the viral cycle. In contrast, only about 10 antigens are produced during the inactive phase: these include the Epstein-Barr virus nuclear antigens (EBNAs 1–6), and the latent membrane proteins (LMPs 1–3).

The heroes in the battle against the Epstein-Barr virus are white blood cells known as cytotoxic T lymphocytes (T-cells) (Box 1: Acquired immunity: Antibodies and T lymphocytes). These cells combine with certain antigens produced by the virus and kill cells that harbour it. Unfortunately, when the virus is associated with Burkitt’s lymphoma and nasopharyngeal carcinoma it appears to produce only one antigen – EBNA1. T-cells are unable to combine with this particular antigen and will not attack the infected cell. In such circumstances, the virus is able to ‘hide’ from the body’s immune system.

The search for a vaccine

As with most viruses, the best chance of defence against Epstein-Barr is vaccination. Yet a vaccine has been elusive, partly because the virus is so good at hiding.

To prevent Burkitt’s lymphoma or nasopharyngeal carcinoma, a vaccine would need to provide 100 per cent immunity or be capable of establishing a T-cell population that recognises EBNA1. Both tasks are extremely difficult. It may be possible, though, to produce a vaccine against glandular fever and post-transplant lymphoproliferative disease, since both produce antigens that are recognised by T-cells.

For several years, Australian scientists have been engaged on a project to produce such a vaccine. Based at the Queensland Institute of Medical Research within the Cooperative Research Centre for Vaccine Technology, they are conducting a trial with a prototype vaccine using human volunteers (Box 2: The future of vaccines).

One of the first steps in the development of the vaccine was to define the antigens of the virus that are important in the immune control of it. Antigens stimulate the manufacture of T-cells: if they – or their most important parts – could be introduced artificially to an individual, then the immune system might be ‘tricked’ into producing T-cells that would recognise the real antigen should infection occur.

The researchers were able to produce a peptide identical to part of the Epstein-Barr virus antigen EBNA3. This peptide forms the basis of the new vaccine: by injecting it into patients, researchers hope to ‘arm’ the body with T-cells in readiness for an invasion of the real disease.

One phase of the trial has been completed; scientists are now confident that the vaccine does not have any harmful effects on patients. The next phase, which is about to begin, will determine the effectiveness of the vaccine in preventing diseases caused by the virus.

Attempts are also being made to grow and expand T-cells in the laboratory to help cure various forms of Epstein-Barr virus-induced cancers. This approach should be particularly useful for treating patients suffering from post-transplant lymphoproliferative disease.

Box 1. Acquired immunity: Antibodies and T lymphocytes

Antibodies

Antibodies are protein molecules that move freely in the bloodstream. They are manufactured by white blood cells in response to the presence of molecules on the surface of the disease-causing microorganism. These molecules are known as antigens. A particular antibody will combine best with the antigen that caused the production of that antibody: thus, the antibody is said to be specific for its antigen. Antibodies react chemically with these antigens in such a way that the microorganism or toxin is neutralised and destroyed before a cell is infected.

T-cells

At the cellular level, the body manufactures what are known as cytotoxic T lymphocytes – T-cells. These white blood cells recognise certain antigens produced by microorganisms and kill cells that harbour them.

If an invading microorganism evades the antibodies and infects a cell, the T-cells will recognise the infected cell and kill it. Thus the two weapons of acquired immunity, the antibody and the T-cell, complement each other.

Thanks for the memory

Once an infection has been overcome, antibodies and T-cells remain in the body, often for years and sometimes for life, acting as a kind of ‘memory’. If the microorganism attacks again, these antibodies ‘remember’ it. They combine with the antigens on the surface of the microorganism and immobilise it so it is no longer able to damage cells in the body. If, despite the efforts of the antibodies, some cells in the body become infected, the memory T-cells are mobilised rapidly to kill the infected cells. Thus, the body has acquired immunity against that particular disease.

Active immunisation

Vaccination works by introducing harmless versions of a disease-causing microorganism or certain antigens of the microorganism to the immune systems of uninfected individuals. This ‘tricks’ the immune system into producing antibodies and, when required, T-cells specific to the microorganism. The body is thus equipped with the right armory should infection by the real microorganism occur. The vaccines used in vaccination are of several types:

• live, attenuated microorganisms;

• killed microorganisms; and

• only one, or a few, antigens of the microorganisms, such as a toxin (toxic molecule) which is first rendered safe (detoxified). This type is called a subunit vaccine and is a type of acellular vaccine.

Passive immunisation

Protection against some infections may be achieved by passive immunisation. With this type of immunisation, antibodies produced in one person are introduced into another. These antibodies are injected into the host shortly before the expected exposure to a disease-causing microorganism. The antibodies are obtained from the blood plasma of people who have had the disease (or have been immunised against it). Injection of the antibodies confers immediate protection against infection for a short time (weeks). It may also be effective if given shortly after such exposure; for example, after being bitten by a snake or rabid dog.

Box 2. The future of vaccines

Gene guns and golden bullets

One such technique uses DNA to produce what are known as nucleic acid vaccines (sometimes called ‘naked DNA’ vaccines). Scientists isolate the genes from disease-causing bacteria or viruses that provide information to make specific antigens. These genes are then inserted into a plasmid (which is a genetic element capable of replicating independently of the chromosomal DNA) which, in turn, is injected into the patient. This can be done in the usual way with a hypodermic syringe into muscle tissue, or with a ‘gene gun’, which fires tiny gold particles coated with the DNA into the surface layers of the skin.

Once inside the body the plasmids penetrate host cells, where they start manufacturing antigens. These antigens, released over a long period, induce an active immune response by the body, including the production of both antibodies and specialised white blood cells (cytotoxic T lymphocytes).

The technique may be used to produce vaccines for both viral and bacterial infections; it shows such promise that clinical investigation of a possible AIDS vaccine has already begun.

‘Antigen factory’ vaccines

A similar technique involves the insertion of selected genes from a disease into benign bacteria or viruses. These are then administered to the patient and serve as a sort of antigen factory, using the inserted genes to churn out antigens of the disease-causing organism. These antigens invoke an immune response that will help protect the patient from a subsequent infection. Several ‘antigen factory’ organisms, including the cowpox virus, are being tested for use in HIV vaccines.

Making vaccines more effective

Conventional vaccines often use weakened or killed cells of the disease itself. While this has proved effective against many diseases, some vaccines developed by this technique produce occasional side-effects in patients. New methods using recombinant DNA technology have led to the development of many vaccines that use only a small part of the disease-causing organism or that use a harmless version of the microbe. For example, by removing genes from the cholera bacterium it becomes safe to use in a vaccine.

These methods produce extremely safe vaccines, but they are often less effective than whole-cell vaccines. A major area of vaccine research seeks ways of making such ‘subunit’ vaccines more effective in producing an immune response. This usually involves the use of what are called adjuvants, which are substances added to the vaccine to aid its operation. Conventional vaccines mostly use aluminium salt as an adjuvant, but recent work has tested oil-based emulsions that contain biodegradable material.

Vaccines have been developed at the University of Oxford in the UK that use genetically engineered viruses as a vector to carry genes for both antigen and adjuvant proteins. This technology could potentially help in the fight against diseases such as malaria and HIV that have been traditionally difficult to vaccinate against.

User-friendly vaccines

Vaccine development agencies recognise the importance of increasing the rate of childhood immunisation. One way of achieving this would be to develop vaccines that could be taken orally or nasally, rather than by injection.

With this in mind, researchers have investigated the use of what are called microcapsules. These consist of an inner reservoir of antigen surrounded by an outer, biodegradable polymer wall, through which the antigen is released slowly. Vaccines administered in this way have been shown to produce strong, sustained immune responses for some antigens. One advantage of microcapsules is that refrigeration is not required, making them suitable for remote regions.

Scientists are currently investigating the possible safety implications of having microcapsules in the body for extended periods. If the method proves to be safe, it may become widely used for vaccine administration.

Combining vaccines

Another way of boosting the rate of childhood immunisation would be to combine vaccines so that patients could be vaccinated against several diseases at one time. Some combinations are already available (the diptheria-tetanus-pertussis vaccine is one example), and researchers continue to seek ways of combining vaccines without reducing their effectiveness.

Linking chains

Armed with an understanding of the molecular structure of antigens for a particular disease, scientists are often able to replicate certain peptides of the antigen in the laboratory. These peptides show promise as vaccines because they can produce an immune response in patients. Indeed, the new Epstein-Barr virus vaccine currently under clinical trial is based on a peptide found on one of the virus’s antigens.

Nevertheless, despite considerable promise, there has been surprisingly little progress in the development of synthetic peptide vaccines. One reason for this might be that the peptides are too small and unstable to provoke an effective immune response.

Australian scientists at the Cooperative Research Centre for Vaccine Technology are pioneering work to polymerise (join together) small peptides. Early results suggest that the polymerisation process aids the potency of the peptides as antigens, and may also allow peptides against more than one disease to be included in the same molecular structure.

Vaccines don’t always target microbe-borne diseases

Trials in the USA have been conducted to test whether drug addiction can be treated with vaccines. Nicotine, cocaine and methamphetamine addiction could all potentially be treated this way. Vaccination for addiction gradually stimulates the immune system to produce antibodies that bind to the drug. This then progressively prevents the drug from entering the brain, reducing the effect on the body with minimal withdrawal symptoms.

The body’s own cancer cells may also be targeted by vaccines. By targeting the specific molecules that are found on the surface of tumour cells, vaccines can produce an immune response to cancerous cells. Cancer vaccines that show promise are those that target telomerase, an enzyme that makes cancer cells immortal.

Related site

• New vaccines help kick drug habits (ScienceDaily, USA)

Activities

• Australasian Science
  • Investigating viruses (Autumn 1996, pages 52-53).

• Access Excellence (USA)
  • Demonstrating An Epidemic – demonstrates the spread of disease.
  • Virus & Bacteria Reports – an activity on viruses and bacteria.

Activity 1. A model of disease transmission

Materials (for each student)

• stock solution in a small test tube
• 1 small test tube
• 1 Pasteur pipette with bulb

Safety notes:

• Do not allow any solution to come into contact with your skin or clothing.

• Notify your teacher immediately if a spill occurs.

• If you splash any solution on yourself, immediately flood the affected area with water.

1. Transfer half of your stock solution to your clean test tube. This will be your solution for exchanges. Think of the solution as aerosol droplets from a sneeze.

2. Round 1: Find one other class member at random and exchange one drop of solution. (The exchange is made by using your Pasteur pipette to place one drop of your solution into that person’s test tube and receiving one drop of their solution in return.)

3. Record the name of your contact in Round 1.

4. Your teacher will signal Round 2. Find a different contact and exchange one drop of solution.

5. Record the name of your contact in Round 2.

6. The teacher will signal Round 3. Find a different contact and exchange one drop of solution.

7. Record the name of your contact in Round 3.

8. Test your solution by adding 1 mL phenol red indicator to your test tube and record the colour. ‘Infected’ solutions are red; all others are yellow.

9. Rinse out all your glassware thoroughly.

10. On the blackboard, record your name, your contacts and whether your solution was ‘infected’ or not.

11. Using the class records on the blackboard, try to trace the transmission route through the class. Attempt to determine the original disease carrier. (You may only be able to narrow it down to two or three individuals.)

12. To establish the original disease carrier, test the stock solutions of the possible disease carriers for the red colour indicating ‘infected’.

Questions

1. What is the maximum number of ‘infected’ individuals possible after three rounds?

2. Why might the observed number be lower than the maximum?

3. What method of disease transmission is not simulated by this model?

4. Suppose that instead of one exchange of solutions each round, you exchanged as many times as you wanted during a specified time period.
   • What differences might be seen in the outcome?

   • How would this change in rules affect your ability to trace the transmission routes?

5. What do public health officials do to help control the spread of infectious diseases?

Teachers notes

Background information

Phenol red is an indicator solution that turns pink or red in alkalis. The ‘infected’ stock solution is 0.1 M sodium hydroxide (NaOH). This concentration should produce a pH that will ensure that even the diluted ‘infected’ solutions will turn red in phenol red. All of the ‘uninfected’ solutions should be yellow in the presence of phenol red. Because the pH of tap water varies, dilute (0.001 M) hydrochloric acid (HCl) is used as the ‘uninfected’ stock solution to ensure ‘uninfected’ samples turn yellow in phenol red. Also ensure that the test tubes do not have residual traces of acids or alkalis.

Preparation

This activity depends on one student in the class having an 'infected' solution that will turn red when phenol red is added. Set up test tubes to be collected so that one tube contains 0.1 M NaOH and all the others contain 0.001 M HCl.

Reagents

• 0.001 M HCl: (CAUTION. ALWAYS ADD ACID TO WATER.) Prepare 1 M HCl by adding 11 mL concentrated (32.3%) HCl to 89 mL water. Prepare 0.001 M HCl by adding 1 mL of 1 M HCl to 99 mL of water. (Prepare enough 0.001 M HCl to give every student (minus one) 2 mL.)

• 0.1 M NaOH: 0.4 grams of NaOH made up to 100 mL with water. (Prepare enough 0.01 M NaOH give 2 mL to at least one student.)

• Phenol red solution: 0.1 grams phenol red dissolved in 100 mL water. (Prepare enough phenol red solution to give each student 2 mL.) Test both stock solutions and diluted samples of the two stock solutions with the phenol red before use.

  If you use the phenol red to test each of their stock-solutions at the end of the third round, you will be able to help students assess any ambiguous results. Use a clean 1 mL pipette to add the phenol red to the students’ test tubes.

Answers to questions

1. A maximum of eight people could be ‘infected’ at the end of three rounds.

2. The number would be lower than the maximum if two ‘infected’ people chose to exchange samples with the same person.

3. Examples of disease transmission not simulated by this model:
   • one infected person sneezing into a room full of people;

   • a single tap infecting a community with cholera.

4. With an unlimited exchange of samples you would expect:

   • there would be many more people ‘infected’ at the end of the round;

   • it would be much more difficult to trace the ‘infection’ route because there would be many more instances of multiple ‘infections’.

5. Public health officials attempt to control infectious diseases by minimising the number of contacts infected people make with the rest of the community by encouraging infected people to stay home. If the disease is serious, infected people are put into isolation wards.

Notes

Make sure that students understand the procedure before beginning.

It is possible to make a number of variations to this activity:

• start with more than one ‘infected’ person in the class;

• allow more than one exchange each round;

• don’t have students record the names of their contacts in each round;

• have only a one-way transfer rather than a two-way exchange.

Activity 2. Diseases caused by the Epstein-Barr virus

• List the diseases caused by the Epstein-Barr virus.

Glandular fever can be transferred from one person to another via saliva, and is classed as an infectious disease.

• Would cancers caused by the Epstein-Barr virus be classed as infectious diseases? Explain your answer.

Teachers notes

Diseases caused by the Epstein-Barr virus include glandular fever, Burkitt’s lymphoma, nasopharyngeal carcinoma, post-transplant lymphoproliferative disease, oral hairy leukoplakia, and possibly some forms of Hodgkin’s disease

Epstein-Barr virus gives rise to cancer in some people but not in others. It appears that if the correct immune response does not develop in an infected individual, then that person is at risk of developing cancer. There is also some evidence that genetic factors play a role in whether or not an infected individual develops cancer.

Students should be able to answer these questions based on material in the key text. You could hand out copies of the key text and set this activity as a small assignment.

Further reading

Useful sites

• EBV Cellular Immunotherapy Project
  http://www.crc-vt.qimr.edu.au/research/passiveimmunity/ebv.html

• Epstein-Barr virus – kiss this disease good-bye
  Provides a list of questions and answers about the Epstein Barr virus and the development of a potential vaccine.
  http://www.crc-vt.qimr.edu.au/media/ebv.html

Glandular fever (Better Health Channel, Australia)

Glandular fever (or kissing disease) is the common term used to describe an acute viral infection called infectious mononucleosis. The virus that causes glandular fever is known as Epstein-Barr virus. Glandular fever mainly affects young adults. A chronic form of glandular fever is one of the suggested causes of chronic fatigue syndrome.
http://www.betterhealth.vic.gov.au/bhcv2/bhcarticles.nsf/pages/Glandular_fever?OpenDocument

The herpes virus (Australian Broadcasting Corporation)

A transcript from The Health Report, 29 July 2002.
http://www.abc.net.au/rn/talks/8.30/helthrpt/stories/s636427.htm

Eureka alert online global news service (American Association for the Advancement of Science, USA)

Provides the latest information on scientific discoveries relating the Epstein Barr virus.
http://search.eurekalert.org/e3/query.html?qt=epstein+barr&col=ev3rel&qc=ev3rel

Mononucleosis (Mayo Clinic, USA)

Infectious mononucleosis (also known as glandular fever) is caused by the Epstein-Barr virus. This clinical description includes symptoms, diagnosis and self-care.
http://www.mayoclinic.com/findinformation/diseasesandconditions/invoke.cfm?id=DS00352

The following sites contain information about infectious mononucleosis and Burkitt's lymphoma:

• Epstein-Barr virus and infectious mononucleosis (Centers for Disease Control and Prevention, USA)
  http://www.cdc.gov/ncidod/diseases/ebv.htm

• Burkitt's lymphoma (Kimball Biology Pages, USA)
  http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/B/BurkittLymphoma.html

The Ras gene and cancer (Access Excellence, USA)

Describes how a class of virus contains an oncogene that causes the development of tumours in humans. A similar situation occurs in Burkitt's lymphoma, but with a different virus and different oncogene.
http://www.accessexcellence.org/AB/BA/Ras_Gene_and_Cancer.html

The following sites offer very technical information about Epstein-Barr virus and the diseases it causes:

• Epstein-Barr virus (Virology Down Under, University of Queensland, Australia)
  Covers the structure of the Epstein-Barr virus, its periodic reactivation, and potential therapeutic treatments.
  http://www.uq.edu.au/vdu/VDUEBV.htm

• Epstein-Barr virus infection: basis of malignancy and potential for therapy (Expert Reviews in Molecular Medicine, 2001, Cambridge University Press, UK)
  Covers the role of Epstein-Barr virus in the development of malignant tumours.
  http://www-ermm.cbcu.cam.ac.uk/01003842h.htm

• Vaccines (Kimball Biology Pages, USA)
  Includes information on different types of vaccines, a table of some of the most widely used vaccines, some problems of vaccine development and DNA vaccines.
  http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/V/Vaccines.html#DNAvaccines

Glossary

antigen. Any foreign substance, usually a protein, that stimulates a specific immune response.

attenuated vaccines. Vaccines are designed to stimulate antibody production without causing serious disease. To make an attenuated vaccine, a disease-causing microorganism is first isolated and then attenuated (made less virulent) by ageing it or altering its growth conditions (such as by depriving it of an essential nutrient). The vaccines for measles, mumps, and rubella are prepared in this way. Because this vaccine is actually a living microbe, it multiplies within your body and therefore causes a strong stimulation of the immune system.

immunity. A body's reaction to the introduction of foreign substances, through the production of defensive substances such as antibodies.

nucleic acid. A large molecule made up of a sequence of phosphorylated nitrogen-containing bases. DNA and RNA are both nucleic acids.

peptide. A molecule consisting of a short chain of amino acids. Longer chains of amino acids are called proteins.

plasmid. A small, circular DNA molecule. Bacteria can have plasmids in addition to the DNA of the main chromosome. Foreign DNA can be added to plasmids. The modified plasmid then transports the DNA into a new cell.

toxins. Substances, produced by microorganisms, which affect the functioning of another organism.