(6) Virology
HONG KONG CENTER FOR HEALTH PROTECTION - Influenza
Flu outbreak in Hong Kong: 6 things you need to know about flu vaccination
WORLD HEALTH ORGANIZATION - Influenza
Turning the tables
It is curiously paradoxical that the prevention of several virus infections was achieved long before anyone knew of the existence of viruses or of the immune responses required to prevent infection. Whereas viruses were first recognized in the 1930s, over 100 years before this, Edward Jenner (1749-1823) succeeded in vaccinating against to induce immunity to severe publish smallpox, the biggest killer virus of all time.
Smallpox prevention and eradication
The first recorded way of preventing smallpox was inoculation, used in China and India for hundreds of years before it reached Western Europe in the 1700s. The technique, also called variolation or engraftment, involved scratching the skin with a needle dipped in scrapings or pus from a smallpox lesion. Unlike virus acquired by inhalation, this generally produced a localized skin infection but no systemic infection and was followed by long-term immunity.
Inoculation was introduced to Britain in the 1720s by Lady Mary Wortley Montagu (1689-1762), who saw it performed while living in Constantinople (now Istanbul) with her husband, Edward Wortley Montagu, the British ambassador to the Ottoman Empire from 1716 to 1717. Lady Mary had suffered from smallpox herself and her brother had died of the disease, so she was willing to try anything that might protect her children. She persuaded the family physician, Dr Charles Maitland, to learn the technique from local practitioners in Constantinople and then inoculate her 5-year-old son Edward.
This he did, and a week later the child developed fever with a few pocks but soon recovered and was then immune.
When the family returned to London in 1718, Lady Mary was keen to publicize inoculation as a way of preventing smallpox, and when an epidemic broke out in 1721, she asked Maitland to inoculate her daughter Mary, aged 4, with two eminent physicians as witnesses. This was successfully accomplished, and the word spread. After further inoculations were carried out on six criminals from Newgate Prison and a group of orphans from London’s Parish of St James without ill effect, King George I gave consent for his two granddaughters to be inoculated, so popularizing the technique.
However, inoculation was bitterly opposed by many of the clergy, who felt that it went against the will of God, and by some doctors who foresaw a loss of income. Others genuinely feared that it might cause smallpox, sparking an epidemic among the non-immune. Indeed, inoculation did sometimes cause full-blown smallpox and had a mortality rate of 1-2%, but this compares with a 10-20%; death rate from smallpox among the un-inoculated. The technique was used widely in Europe and the USA until the safer method of vaccination was introduced at the beginning of the 19th century.
Edward Jenner was a country doctor from Berkeley, Gloucestershire, UK, where it was rumored that milkmaids’ unblemished skin was due to contracting cowpox, a natural infection of cows’ udders, and thereafter being immune from smallpox. These rumors possibly stemmed from Benjamin Jesty (1736-1816), a farmer from Dorset, who was probably the first to test this theory, in 1774, when he inoculated his wife and children with cowpox, but he did not pursue the experiment any further. It is not clear whether Jenner knew of Jesty’s work before he decided to test the theory for himself, but he later acknowledged Jesty’s contribution.
Jenner went for the most direct proof possible. In his now world-famous experiments, which by today’s standards would not proceed on ethical grounds, he obtained cowpox from the arm of an infected milkmaid, Sarah Nelmes, and used it to inoculate a child, James Phipps, who had not had smallpox. A few weeks later, he inoculated Phipps with live smallpox to see if he was protected.
Fortunately, Phipps remained healthy, and when several other children tested with cowpox were also protected from smallpox, Jenner knew he had made a groundbreaking discovery that had the potential to save many thousands of lives.
However, when Jenner published his findings in a pamphlet in 1798, Archaeology At first, cowpox virus for vaccination was obtained from naturally infected cows or milkmaids, but arm-to-arm passage from inoculated to non-immune people was soon developed, and later the virus was grown on, and harvested from, the flanks of cows, a method more suited to large-scale production.
The practice of vaccination remains almost unchanged to the present day and was essential for worldwide smallpox eradication.
By 1966, when the WHO announced the Smallpox Eradication Campaign, the virus had already been eliminated from Europe and the USA, but was still endemic in 31 countries, giving an estimated 10 million cases and 2 million deaths annually. The campaign was predicted to be costly, but as the disease was so deadly, even countries that had eliminated the virus lived in fear of imported cases causing an epidemic and so were willing to provide funds for global eradication.
The success of this bold, highly complex, and expensive endeavor critically depended on several specific features of the smallpox virus, the disease itself, and the vaccine. Firstly, the virus has no animal reservoir; it only infects humans, causing an acute illness with no virus persistence in survivors. So as the virus has nowhere to hide, interruption of its chain of infection should eventually lead to its elimination. Secondly, that this disease was non-infectious until the symptoms appeared, when they were severe enough to keep the patient relatively isolated in bed.
The disease itself was easy to diagnose from the clinical features, particularly the characteristic rash. So since no silent infections occurred, virtually all cases could be identified and isolated. Furthermore, the incubation period of around two weeks provided a window of opportunity for chasing the contacts of a case and isolating them until they were deemed non-infectious. Thirdly, that the vaccine, which was absolutely key to the success of the campaign, was safe and highly effective. And as smallpox virus is a stable DNA virus with only the one major type, there was little likelihood of it mutating into a vaccine resistant strain.
A vaccine preparation that remained active in tropical climates was produced and distributed by armies of workers in the world’s four remaining endemic zones: Brazil, Indonesia, sub-Saharan Africa, and the Indian subcontinent. The aim was to increase vaccination coverage to over 80%;, the critical level for preventing virus spread. This worked so well that within 10 years smallpox transmission was finally interrupted, Ethiopia being the last endemic country. Worldwide, eradication of smallpox was declared in 1980.
Amazingly, the last two cases of smallpox worldwide occurred in the UK in 1978. These were related to ongoing smallpox virus research in the Depart of Microbiology at the University of Birmingham Medical School where one victim, a photographer in the Anatomy Department, died and another who caught the disease from her recovered. The Anatomy Department was situated on the floor above the microbiology laboratories, and the enquiry that followed the disaster found that the conditions used to contain the virus in the laboratory were ‘far from satisfactory’.
The report suggested that the virus had travelled via air ducts from the virus preparation area to a phone box in the Anatomy Department on the floor above that was often used by the photographer. The whole incident had a final upsetting outcome when the Head of the Microbiology Department committed suicide following the enquiry’s highly critical report of the Department’s safety procedures.
Jenner’s vaccine works by generating an immune response to a harmless virus (cowpox) that is so closely related to the lethal virus (smallpox) that the immune system cannot distinguish between the two. This same trick was later used to prevent Marek’s disease, a devastating infection of poultry caused by a tumor-associated herpes virus called Marek’s disease virus. It mainly affects chickens and rapidly kills up to 80%; of a domestic flock, causing severe financial loss. The disease, first described by Hungarian pathologist Jozef Marek (1868-1952) in 1907, begins with paralysis of one or more limbs followed by difficulty in breathing leading to death. These symptoms are caused by T cells infiltrating the nerves and producing tumors in vital organs. Once the virus was isolated in 1967, it was soon discovered that a very similar virus, herpes virus of turkeys, could protect chickens from Marek’s disease virus without ill effect.
Rabies vaccination
Several years after Jenner’s experiments, Louis Pasteur, working in Paris, made a vaccine against rabies virus from dried spinal cords of rabies-infected animals. This virus is present in saliva from rabid animals and generally circulates among wild animals such as dogs, foxes, and bats. Although some species can survive an attack of rabies, untreated human infections, usually acquired through the bite of a rabid dog, are 100%; fatal. Death results from the virus invading the brain, but not before it has induced the most distressing symptoms. These include the classic hydrophobia (fear of water) combined with periods of extreme excitement and hyper-activity interspersed by lucid intervals when the patient is all too aware of their desperate plight. Patients experience terrifying spasms of their respiratory muscles when trying to drink, but thirst drives them to repeated attempts to drink, with violent effects that may lead to generalized convulsions and cardiac or respiratory arrest. Otherwise, patients survive in this state for about a week before sinking into a coma and dying. It is no wondered that Pasteur chose rabies as the first infectious disease he attempted to prevent with a vaccine.
In 1885, while his vaccine was still being tested in the laboratory, Pasteur was persuaded to try it out on a child, Joseph Meister, who had been badly bitten by a rabid dog and whose outlook was grim. The vaccine saved the child’s life, and many others thereafter, until it was replaced by a safer preparation made by growing the virus in cultured cells.
Unlike vaccines designed to prevent acute infections such as measles and polio, rabies vaccine can protect from the disease even if it is given after the bite that transmits the virus. This is known as ‘post-exposure’ vaccination. This is because the virus must follow nerve pathways from the site of infection to the brain before causing symptoms.
There is no doubt that although vaccines are expensive to prepare and test, they are the safest, easiest, and most cost-effective way of controlling infectious diseases worldwide. For this reason, vaccines against almost every pathogenic virus from the common cold virus to the highly lethal Ebola virus are currently in preparation. But vaccine development is a long-drawn-out process, and although several are in clinical trials, relatively few have been licensed for clinical use. These include a triple vaccine for the once common childhood illnesses, measles, mumps, and rubella, given by two injections, one at 13 months and one at 3 to 5 years of age.
Traditionally, there are two types of viral vaccines, one using live attenuated (weakened) virus and the other inactivated virus. The pros and cons of using these different vaccines are illustrated by the story of polio eradication, which has now entered its end game.
During the early 1900s, polio was a much-feared disease. Epidemics reached a peak in the USA in the 1950s, just before the inactivated vaccine produced by American virologist Jonas Salk (1914-1995) came into use. It had an immediate effect, reducing the number of polio cases in the USA from 20,000 to around 2,000 per year. However, it had to be given by injection, and at first it was of fairly low potency.
For these reasons, another American virologist, Albert Sabin (1906-1993), manufactured a live attenuated polio vaccine that became available in the early 1960s. He grew the virus in the laboratory until a weakened strain emerged that induced immunity without causing disease. This vaccine was cheaper and easier to produce than the inactivated product and could be taken orally, a great advantage, particularly for use in the developing world. Furthermore, oral administration uses the natural route of wild polio virus infection, and so the vaccine strain replicates in the gut and is excreted in faeces. It can then spread in the community, effectively vaccinating those who have not officially received a dose of vaccine. However, because the virus grows in the body, there is a chance that it will mutate into a pathogenic strain. Although rare, this does occur, with live attenuated polio vaccine causing paralytic polio in about one in a million vaccines.
The WHO Polio World Eradication Campaign begun in 1988 aimed to achieve over 80%; coverage with oral vaccine. This was highly successful in eradicating wild virus, and the global incidence had declined by 99%; by 2005, with just a few pockets of infection remaining in Afghanistan, India, Pakistan, and Nigeria. Paradoxically, as the incidence of wild polio infection declined, the relative risk of vaccine-related polio caused by mutant vaccine virus rose, so that now most cases of paralytic polio are caused by the vaccine strain. Also, with the vaccine strain of polio virus circulating in the community, it is not possible to completely eradicate the virus. For these reasons, several Western countries have reverted to using the inactivated vaccine, and this will probably have to happen worldwide before complete eradication can be achieved.
Other human viruses on the list for worldwide eradication include measles, rubella, around 5,000 to 10,000 years ago, re0Smumps, rabies, and HBV.
To vaccinate or not to vaccinate?
The ethical debate surrounding the use of smallpox vaccination in Jenner’s time has moved on but certainly not disappeared. There are still religious sects who refuse vaccination, but other major issues have now come to the fore.
One of these is the ‘hygiene hypothesis’ invoked to explain the recent rise in autoimmune and allergic diseases in Western countries. Both these types of disease are caused by an imbalance in the immune response. The hygiene theory attributes this to a lack of childhood infections resulting from vaccinations as well as rising standards of hygiene and antibiotic use in the modern world. All these factors decrease antigenic stimulation during childhood and could predispose a child’s immune system to these abnormal responses. Research in this field continues, but at the time of writing, there is no concrete evidence to support the hypothesis.
However safe vaccines are, they will never be completely without potential side effects. As they continue to succeed in preventing infectious diseases, so death rates will fall, and eventually the adverse effects of a vaccine may exceed those of the disease it was designed to prevent. Although the risks of smallpox vaccine are exceedingly small, at one or two deaths per million vaccinations, this was bound to happen at some point during the smallpox eradication programme as the virus was banished from whole continents. Even so, it was still essential that vaccination continued until complete eradication was ensured. At the present time, while global eradication of measles is ongoing, and infection is now a rare event in the developed world, some may think that with a one in a million risk of vaccine-associated encephalitis, it is safer not to vaccinate. However, if enough people argue this way and the level of vaccination falls below the critical level of 80%;, then measles epidemics will reappear, leading inevitably to deaths.
This is exactly what happened in the UK after a report appeared in the medical journal The Lancet, in 1998 suggesting a link between measles vaccination and childhood autism. The publicity this received caused an immediate downturn in measles vaccinations and, despite the link being refuted and eventually disproved, the dip lasted long enough for the virus to re-establish itself in the community and cause measles epidemics and deaths. It took 12 years for the report’s senior author, Andrew Wakefield, to be found guilty of dishonesty and flouting ethics protocols by the General Medical Council and to be struck off the UK Medical Register. Only then did The Lancet, officially retract the report on the basis of false claims of ethical approval.
For all these reasons, there is a constant search for safer vaccines. The molecular revolution beginning in the 1960s heralded a new generation of recombinant subunit viral vaccines. With the molecular makeup of viruses finally unraveled, the key viral proteins (subunits) required to stimulate protective immunity could be identified and manufactured in the laboratory as a vaccine. The first of these new recombinant vaccines to come on line was against HBV. HBV surface antigen was identified as the key protein, and this was cloned and produced in vast quantities in yeast cells in the laboratory. After animal experiments showed the vaccine to be safe and effective, it replaced earlier products made by purifying HBV surface antigen from the blood of persistently infected individuals, a practice that carried the risk of also transferring blood-borne infections such as HIV and HCV. A similar laboratory-based product is the recently licensed vaccine against the cancer-causing HPV types 16 and around 5,000 to 10,000 years ago, re0S18 based on the major viral coat protein. These HPV protein molecules are assembled into hollow, non-infectious ‘virus-like particles’ that have been shown, using animal models, to be safe and to prevent HPV-induced cancer development. The vaccine is now recommended for teenage girls to prevent cancer of the cervix.
Other modern inventions using recombinant vaccines are so-called naked DNA vaccines, for which the DNA that codes for the key viral protein is either injected directly or inserted into the genome of a harmless virus for delivery. When this virus, called a vector, infects human or animal cells, it expresses the key ‘foreign’ gene along with its own genes and generates a host immune response. No vaccines made in this way have yet been granted a license for human use, but clinical trials have been carried out on a recombinant HIV vaccine using an adenovirus as a vector.
Despite these varied approaches to vaccine production, there are still many pathogenic viruses with no available vaccines, including the childhood killer respiratory syncytial virus. There are a variety of reasons for this, which are illustrated by the many failed attempts to prepare a vaccine against HIV.
HIV vaccines: fact or fiction?
It is now over 20 years since HIV was first identified as the cause of AIDS, but despite massive financial investment and scientific effort, there is no effective vaccine on the horizon. After HIV vaccine preparations that primarily stimulated antibody responses failed to prevent infection, T cell vaccines were tried, but these too have failed - one even seemed to increase infection rates in the vaccinated group compared to the controls.
There are several reasons for these failures. Firstly, HIV mutates rapidly, and after around 100 years of human infection there are many different types and strains that may not all be prevented by a single vaccine preparation. Secondly, HIV persists in everyone it infects, indicating that the natural immune response against it cannot clear the virus. This makes it tough to design a vaccine that will do what nature cannot achieve. Thirdly, HIV is usually transmitted via the lining of the genital tract, so antibodies and immune T cells in the blood must reach this site to prevent HIV infecting CD4 cells and establishing latent infection. Finally, HIV may be transmitted either as free virus or inside cells such that the immune response required to prevent it establishing infection in each case may be different. For all these reasons, the ideal vaccine that prevents HIV infection entirely is at present a remote possibility. Even a vaccine that controls the infection and prolongs the disease-free period would be helpful. A slight glimmer of hope came in 2009 when the results of the largest and most expensive HIV vaccine trial to date were announced. The trial, involving 16,000 volunteers in Thailand, took six years to complete and was generally expected to fail. However, the results showed a modest level of protection produced by two recombinant vaccines given in a ‘prime boost’ scenario.
The first shot was designed to stimulate a T cell response to HIV and the second to boost this response. Even if this is the beginning of a breakthrough for HIV vaccines, a licensed product is still a long way off. In the meantime, other ways of tackling the deadly infection must be used to maximum effect. The multifaceted approach to controlling HIV still focuses on interrupting spread of the virus but goes beyond the traditional means of education, free access to condoms, and needle-exchange programmes and prompt treatment of other sexually transmitted infections. For instance, circumcision has been shown to reduce the risk of infection in men from person to person–0S by 40–80%; and is therefore to be encouraged in certain high-risk groups.
Antiviral drugs are key in curtailing viral spread and are being rolled out worldwide, with present coverage of around 50%; of those in need. A priority area is to deliver antiretroviral drugs to all HIV positive pregnant women to prevent virus transfer to the child, and this is expected to be implemented by 2015.
Taking a leaf out of the malaria prevention book, where pre-exposure prophylaxis is the norm, one option is to protect high-risk, uninfected partners of HIV-infected people with antiretroviral drugs. In addition, the post-exposure prophylaxis used successfully in healthcare workers after accidental occupational HIV exposure is an option following high-risk sexual encounters, mirroring the thinking behind the night-after contraceptive pill.
Many studies show that HIV transmission occurs most readily when the viral load in the blood is high, and since antiviral therapy can reduce this load to undetectable levels, these drugs can be used to prevent spread. Most transmission occurs in the few months following primary infection when the viral load is extremely high but when most people are unaware of their infection. More effective screening programmes of at-risk groups, including opt-out testing, would pick up these early infections and allow early treatment.
Antiviral agents
For almost 40 years after the discovery of penicillin in 1945, bacterial infections could be cured with the appropriate antibiotic, while most virus infections were untreatable. This contrast relates to the biological differences between bacteria and viruses and in the way they cause disease. Pathogenic bacteria are mostly free-living, single-celled organisms that can invade and multiply in the body, so causing disease. Bacteria have tough outer cell walls that are essential for their survival, and penicillin and its derivatives target these unique structures while leaving host cells unharmed. However, viruses are not cells, and because they use the replication machinery of the cells they infect, it has proved difficult to find drugs that prevent virus replication without damaging the host. Despite this, there are now almost 40 antiviral drugs approved for clinical use. Unfortunately, most are only active against a single virus or virus group.
The first antiviral drug to be licensed was aciclovir, made in the 1970s and active against herpes virus infections such as cold sores and shingles. The drug masquerades as a nucleoside, the building block of DNA. In order to be incorporated into herpes virus DNA, phosphate groups must be added to each nucleoside by a herpes virus enzyme called thymidine kinase. This essential step restricts the drug’s activity to virus-infected cells. Phosphorylated aciclovir then joins the growing viral DNA chain and blocks its extension, so terminating viral DNA replication. By targeting a virus-specific function, in this case replication of its DNA, aciclovir spares uninfected host cells and therefore does no collateral damage.
The recognition of HIV as the cause of AIDS in the early 1980s gave a much needed impetus to antiviral drug discovery. Now around half of the licensed antiretroviral compounds are specifically designed for HIV treatment and have transformed a uniformly fatal infection into a chronic disease.
Many antiretroviral compounds act in a similar way to aciclovir by inhibiting viral enzymes essential for viral replication, in this case targeting HIV’s reverse transcriptase, protease, or integrase enzymes. Other drugs inhibit HIV’s entry into cells. But since HIV mutates frequently, it rapidly generates resistance to a single drug.
Flu is another infection that can be treated with a variety of antiviral drugs. These are based on two modes of action: one inhibits the virus’s neuraminidase enzyme and the other blocks virus entry into host cells. During the short course of treatment required to cure flu, drug resistance is not generally a problem, but in an epidemic or pandemic situation it may be. As we saw in the 2009 H1N1 swine flu pandemic, the drug Tamiflu (Oseltamivir, which is a neuraminidase inhibitor) was stockpiled by many governments in developed countries. This worked fine at the beginning of the pandemic, but then resistant strains began to circulate. The hope was that the drug would fill the gap while a vaccine was prepared. This approach worked reasonably well, particularly for severe cases. However, since the pandemic flu strain turned out to be generally mild, the strategy was not really put to the test.
Clearance of persistent hepatitis viruses
On a worldwide scale, persistent HBV and HCV are an enormous problem, accounting for around 250,000 deaths annually. And yet some people clear these viruses after primary infection, so treatment aims to induce clearance in those who suffer persistent active infection. At present, this is not always possible, but the combination of antiviral drugs and immune stimulants can often suppress virus replication and restrict liver damage.
The cytokine interferon-α has both immune-stimulating and antiviral effects and is used for treatment of both viruses. However, there is a serious downside. The treatment involves a long course of injections with some unpleasant side effects, mostly flu-like symptoms with lethargy. It also sometimes causes depression, and around 15%; of patients are unable to complete the course. Used as a single therapy, interferon-a gives a sustained response in up to 40% of people with persistent HBV infection, and similar results are obtained with single antiviral drugs. The latter are presently the treatment of choice, but clinical trials are in progress to assess the role of combining interferon-a with antiviral drugs for HBV management. Persistent HCV also responds to interferon-α and a response rate of up to 80%; is obtained when this is combined with antiviral drugs. The outcome depends on infecting HCV subtype, the extent of the disease, and the age and sex of the patient. The best results are achieved in individuals with subtypes 2, 3, or 4 and a low viral load.
Virus diagnosis
Historically, diagnosis and treatment of virus infections have lagged far behind those of bacterial diseases and are only now catching up. Originally, viruses were identified as infectious agents that passed through filters with a pore size small enough to trap bacteria. Then in the 1930s, the invention of the electron microscope allowed visualization of viruses and led to resolution of their structure and an understanding of their life cycaccine is chea.
