Stephen Korsman

Vaccines are apathogenic entities that cause the immune system to respond in such a way, that when it encounters the specific pathogen represented by the vaccine, it is able to recognise it - and mount a protective immune response, even though the body may not have encountered that particular pathogen before.

Influenza viruses have been with mankind for at least 300 years, causing epidemics every few years and pandemics every few decades. They result in 250,000 - 500,000 deaths, and about 3-5 million cases of severe illness each year worldwide, with 5-15 % of the total population becoming infected (WHO 2003). Today, we have the capability to produce 300 million doses of trivalent vaccine per year - enough for current epidemics in the Western world, but insufficient for coping with a pandemic (Fedson 2005).

The influenza vaccine is effective in preventing disease and death, especially in high risk groups, and in the context of routine vaccination, the World Health Organization reports that the "influenza vaccine is the most effective preventive measure available" (WHO 2005e). With regard to the present fear of an imminent influenza pandemic, "Vaccination and the use of antiviral drugs are two of the most important response measures for reducing morbidity and mortality during a pandemic." (WHO 2005d).

The different types of vaccines in use today for influenza can be divided into killed virus vaccines and live virus vaccines. Other vaccines of these two types are under development, as well as some that do not fall into either category, where a degree of genetic manipulation is involved.

Killed virus vaccines can be divided into whole virus vaccines, and split or subunit vaccines.

Whole virus vaccines are safe and well tolerated, with an efficacy of 60-90 % in children and adults.

Split vaccines are produced in the same way as whole virus vaccines, but virus particles are disrupted using detergents, or, in the past, ether.

Subunit vaccines consist of purified HA and NA proteins, with the other viral components removed. Split and subunit vaccines cause fewer local reactions than whole virus vaccines, and a single dose produces adequate antibody levels in a population exposed to similar viruses (Couch 1997, Hilleman 2002, Potter 2004). However, this might not be sufficient if a novel pandemic influenza virus emerges, and it is believed that two doses will be required.

Inactivated influenza virus vaccines are generally administered intramuscularly, although intradermal (Belshe 2004, Cooper 2004, Kenney 2004) and intranasal (mucosal) routes (Langley 2005) are being investigated.

Cold-adapted live attenuated influenza virus (CAIV) vaccines, for intranasal administration, have been available in the USA since July 2003, and in the former Soviet Union, live attenuated influenza vaccines have been in use for several years. The vaccine consists of a master attenuated virus into which the HA and NA genes have been inserted. The master viruses used are A/Ann Arbor/6/60 (H2N2) and B/Ann Arbor/1/66 (Hoffman 2005, Palese 1997, Potter 2004). The vaccine master virus is cold-adapted - in other words, it has been adapted to grow ideally at 25 degrees Celsius, which means that at normal human body temperature, it is attenuated. The adaptation process has been shown to have caused stable mutations in the three polymerase genes of the virus, namely PA, PB1, and PB2 (Hilleman 2002, Potter 2004).

The advantages of a live virus vaccine applied to the nasal mucosa are the development of local neutralising immunity, the development of a cell-mediated immune response, and a cross-reactive and longer lasting immune response (Couch 1997).

Of concern in the CAIV vaccine, is the use in immunocompromised patients (safety ?) and the possible interference between viral strains present in the vaccine which might result in decreased effectiveness. Damage to mucosal surfaces, while far less than with wild-type virulent influenza viruses, may lead to susceptibility to secondary infections. Safety issues, however, do not seem to be a problem in immunocompetent individuals. Of greater concern for the future is the possibility of genetic reversion - where the mutations causing attenuation change back to their wild-type state - and reassortment with wild-type influenza viruses, resulting in a new strain. However, studies done to test for this have not detected problems so far (Youngner 1994).

It is hoped that cell culture, using Madin-Darby Canine Kidney (MDCK) or Vero (African green monkey kidney) cells approved for human vaccine production, may eventually replace the use of hens' eggs, resulting in a greater production capacity, and a less labor-intensive culturing process. However, setting up such a facility takes time and is costly, and most vaccine producers are only now beginning this process.

Reverse genetics allows for specific manipulation of the influenza genome, exchanging genome segments for those desired (Palase 1997, Palese 2002b). Based on this method, several plasmid-based methods (Neumann 2005) for constructing new viruses for vaccines have been developed, but are not yet in use commercially. A number of plasmids, small circular pieces of DNA, containing the genes and promoter regions of the influenza virus, are transfected into cells, which are then capable of producing the viral genome segments and proteins to form a new viral particle. If this method could be used on a larger scale, it may simplify and speed up the development of new vaccines - instead of the cumbersome task, for the live attenuated vaccines, of allowing reassortment in eggs, and then searching for the correct reassortment (6 genes from the vaccine master strain, and HA and NA from the selected strain for the new vaccine), the vaccine producers could simply insert the HA and NA genes into a plasmid.

DNA vaccines have been tested for a variety of viral and bacterial pathogens. The principle upon which the vaccine works is inoculation with DNA, which is taken up by antigen presenting cells, allowing them to produce viral proteins in their cytosol. These are then detected by the immune system, resulting in both a humoral and cellular immune response (Hilleman 2002).

Vaccines to conserved proteins have been considered, and among the candidates are the M2 and the NP proteins. It is hoped that, by producing immunity to conserved proteins, i.e. proteins that do not undergo antigenic change like HA and NA do, a vaccine can be produced that does not need to be "reinvented" each year. This is also on the WHO's agenda for a pandemic vaccine (Couch 2005). Such vaccines have been shown to be effective in laboratory animals, but data are not available for human studies. "Generic" HA-based vaccines, aimed at conserved areas in the protein, are also being considered (Palese 2002b).

Adjuvants have been used in a number of vaccines against other pathogens, and are being investigated for a role in influenza vaccines. The purpose of adjuvants is to increase the immune response to the vaccine, thus allowing either a decrease in antigen dose, a greater efficacy, or both. Alum is the only adjuvant registered in the United States, and MF59, an oil/water emulsion, has been used in influenza vaccines in Europe since 1997 (Wadman 2005). A vaccine using the outer membrane proteins of Neisseria meningitidis as an adjuvant has shown success in early clinical trials (Langley 2005).

Replication-defective influenza viruses can be made by deleting the M2 or the NS2 genes (Hilleman 2002, Palese 2002b). Only a single round of replication can occur, with termination before the formation of infectious viral particles. Protein expression will result in an immune response, and there is no danger of infection spreading to other cells or people.

Antibody response, determined by measuring haemagglutination inhibition titers, is used as a serological marker of the immunological response to the vaccine, or efficacy. In persons primed by previous exposure to viruses of the same subtype, antibody response is similar for the various types of vaccines. However, in persons without such previous exposure (either through vaccination or through natural infection), response is poorer in the split and subunit vaccines, where two doses are required.

In healthy primed adults, efficacy after one dose ranges from 80-100 %, while in unprimed adults, efficacy enters into this range after two doses. In other populations, efficacy is lower:

*adapted from Pirofzki 1998, Potter 2004, Musana 2004

Effectiveness, usually defined by prevention of illness, is generally slightly lower, with 70-90 % effectiveness in children and healthy adults under the age of 65. In those above 65 years of age, a lower rate of 30-40 % is seen. However, the vaccine is 20-80 % effective in preventing death from influenza in persons older than 65 years, with revaccination each year reducing mortality risk more than a single vaccination (Govaert 1994, Gross 1995, Nichol 1994, Partriarca 1985, Voordouw 2004). In patients with previous myocardial infarctions (MI), a study by Gurfinkel et al. (2004) showed a reduction in the one year risk of death (6 % in the vaccinated group, 13 % in the control group) and combination of death, repeat MI, or rehospitalisation (22 % versus 37 %), possibly due to a non-specific effect of immune responsiveness. Further studies are planned to evaluate the impact of influenza vaccination on acute coronary syndromes.

Vaccination of caregivers against influenza also reduces the exposure of vulnerable populations to influenza.

Studies have been done on effectiveness in terms of health benefits and cost in several healthy populations (Bridges 2000, Langley 2004, Monto 2000, Wilde 1999). They suggest that, while individual health benefits from vaccination certainly exist, as do reductions in days absent from work, vaccinating healthy working adults may not provide cost savings when compared to loss of productivity and days taken off due to illness. Vaccinating health care professionals is recommended, not only because of health benefits and reduced days absent from work, but because it is believed that hospital employees tend to report to work in spite of having an acute febrile illness. Previous studies have shown that vaccinating health care professionals reduces nursing home and hospital-acquired influenza infections (Pachuki 1989, Potter 1997).

Guillain-Barré Syndrome is seen as the most dangerous side effect of influenza vaccines, aside from manifestations of egg allergy. It is, however, rare: the annual reporting rate decreased from a high of 0.17 per 100,000 vaccinees in 1993-1994 to 0.04 in 2002-2003 (Haber 2005).

The most frequent side effects are pain, redness, and swelling at the injection site (10-64 %) lasting 1-2 days, and systemic side effects such as headache, fever, malaise, and myalgia in about 5 % of vaccinees (Belshe 2005, Musana 2004, Potter 2004). These side effects are largely due to a local immune response, with interferon production leading to systemic effects. Local side effects are more common with whole virus vaccines than subunit or split vaccines, and also more common with intradermal vaccination than intramuscular vaccination.

Since the inactivated vaccines do not contain live virus, they cannot cause influenza infection - often respiratory illness is incorrectly attributed to influenza vaccination. Live attenuated virus vaccines do contain live virus; however, side effects are rare, with a runny nose, congestion, sore throat, and headache being the most commonly reported symptoms, with occasional abdominal pain, vomiting, and myalgia (Musana 2004). They are not recommended for use in children below the age of 5 years, although a study by Piedra et al. (Piedra 2005) showed safety in children above the age of 18 months. Controversies have arisen around the possibility of exacerbated asthma in children between 18-34 months of age (Bergen 2004, Black 2004, Glezen 2004). It should be noted, however, that these vaccines should be avoided in immunocompromized patients.

http://www.fda.gov/cber/flu/flu.htm

Table 2 shows some of the available influenza vaccines, with links to FDA and package insert data.

*FluMist is the only currently available live attenuated influenza vaccine. All others are inactivated.

Several methods of reducing the amount of antigen in vaccine preparations have been investigated. Most importantly are the use of adjuvants and the exploitation of a part of the immune system designed to elicit an immune response - dendritic cells.

Adjuvants are used in a number of vaccines in current use, such as those for Diphtheria/Tetanus/Pertussis (DtaP) and Haemophilus influenzae (Hib). Examples of adjuvants include alum (a combination of aluminum compounds), liposomes, emulsions such as MF59, Neisseria meningitidis capsule proteins, immunostimulating complexes (ISCOMs), and interleukin-2. They enhance the immune response to a vaccine, allowing a lower dose to be given, while maintaining sufficient protective response (Couch 1997, Langley 2005, Potter 2004).

In the event of a shortage of vaccine, as happened in the 2004/5 influenza season, as well as in the event of a pandemic situation, certain individuals, such as those working in the healthcare sector and in the poultry industry, and those exposed on the front lines, will need to be given priority over other groups for access to vaccines. As has happened in the past, leaders may have identify groups for urgent vaccination in order to allow for maximum functioning of essential services, while other groups may have to wait until a greater supply is available (MacReady 2005, Treanor 2004). In the event of a pandemic, this could become problematic, but recent experience in the 2004/5 shortage showed that it was managed well by most (Lee 2004), with some instances of companies buying up vaccine, leaving private practices and public health services without supply (MacReady 2005). In the UK, there have already been debates about who should get the H5N1 pandemic vaccine first - healthcare workers, or poultry workers - if H5N1 avian influenza were to reach Britain (Day 2005).

The purpose of this section is not to be an exhaustive reference on avian influenza vaccine development. That is a rapidly advancing field, and the achievements of those involved will likely change the face of influenza vaccinology, and vaccinology in general. In 10 years from now, it is likely that we will look back on our current influenza vaccines and think of them as primitive. Details and advances noted now will be outdated tomorrow. This section will provide an outline of the current direction, the problems we face at the moment, and where we can hope to be in the near future.

As we have seen, vaccination against influenza is a crucial weapon, not only in our fight against seasonal influenza, but against a pandemic that may come tomorrow, next year, or in the next decade. We need to prepare ourselves now.

The World Health Organisation is working with leaders of countries and vaccine manufacturers around the world to prepare for the pandemic many fear will arise out of the current H5N1 avian influenza scare (WHO 2005g).

Although it is an ongoing process, initial strains of H5 avian influenza, such as A/Duck/Singapore/97 (H5N3), have been identified for use in vaccine development (Stephenson 2005). However, it should be noted that the focus is not solely on H5 strains - H2, H6, H7, and H9 are not being ignored, although only H1, H2, H3, N1 and N2 have been found in human influenza viruses (Kilbourne 1997).

Our most urgent needs are a) a stockpile of anti-influenza drugs, b) a vaccine that matches the pandemic strain, c) expedited testing and approval of this vaccine, and d) the capacity to mass-produce enough vaccine to provide the world with a good defense. At present, all of these are still in their infancy.

A matching vaccine will require knowledge of the pandemic strain, and until the next pandemic begins, we will not know for certain what that strain will be. Current efforts are working with a number of strains, mostly H5 strains, as this seems to be the most likely origin at the present time.

The technology to rapidly develop such a vaccine needs to be fully developed. At present, there are several methods being used to develop candidate vaccines.

• Cell culture systems, using Vero or MDCK cell lines, are in development, and will increase our production capacity. The cells could be grown on microcarriers - glass beads - to enable high volume culture (Osterholm 2005). However, these will take several years to put in place, and the cost is problematic (Fedson 2005).
• Reverse genetics is being used to design candidate vaccines - for example, H5N1 virulence genes have been removed from a laboratory strain. Attenuating the virulence of the virus is important, considering the increased mortality rate of the current highly pathogenic H5N1 avian influenza when it does enter human hosts. While the H5N1 mortality rate in humans at present doesn't necessarily reflect the mortality rate in an eventual pandemic, serious attention must be paid to the pathogenicity of the current H5N1 strain before it can be used in a vaccine.
• Plasmid systems are in development - several exist, and others are being described in the scientific literature. A generic influenza virus would supply 6 genes in plasmid form, and once the pandemic strain is identified, it would supply the HA and NA genes. DNA vaccine development experiencing a limited success.
• Apathogenic H5N3 with an adjuvant is being tested - the immune response will be against the H5 only, but the important aspect here is the use of an attenuated strain (Horimoto 2001).
• Live attenuated cold adapted virus is being considered. This may open even more doors for potential reassortment, however, and it may take considerable time to demonstrate safety in certain populations, such as the elderly and children.
• H5N2 inactivated vaccines exist for poultry, and appeared to be protective against H5N1 from 2002 and 2004, but it is expected that human vaccines will have to be better matched than poultry vaccines (Lipatov 2004).

In order to ensure that, when the time comes, a vaccine can be rapidly produced, tested, and shown to be safe, immunogenic, and protective, the WHO has asked vaccine manufacturers and scientists to start developing new vaccines based on strains that may be related to an eventual pandemic strain. These vaccines will likely never be used, and are being developed to demonstrate that when the actual pandemic vaccine is needed, the principle is sound, and the technology is in place and proven on previous vaccines - hence the term "mock vaccine". The important aspect is the development of established vaccines that do not need lengthy studies before they can enter the market. They need to contain viral antigens humans have not had previous exposure to, such as the H5N1 antigens, and companies need to take them through clinical trials to determine immunogenicity, dose, and safety, and ultimately be licensed for use in the same stringent procedures used for other vaccines.

Currently, an expedited system is in place for the inactivated influenza vaccines against seasonal human influenza - the whole process, from the identification of the strains to be used, to the injection in the consultation room, takes about 6-8 months, because the vaccine is an established one, and only certain aspects need to be confirmed prior to release. This same system needs to be in place for a pandemic vaccine (Fedson 2005, WHO 2004a-b).

In an ideal world, 12 billion doses of monovalent vaccine would be available in order to administer two doses of vaccine to every living human being.

The reality is that we do not have this much available.

Currently, the world's vaccine production capacity is for 300 million doses of trivalent vaccine per year. This amounts to 900 million doses of monovalent vaccine, if all production were shifted to make a pandemic vaccine. Considering that at least two doses will be needed, the current capacity serves to provide for only 450 million people. This is further complicated by the fact that the dose of antigen that will be required is not yet known, but studies indicate that it may be higher than current human influenza vaccines (Fedson 2005).

The world has suffered from vaccine shortages before - recently in the 2004/5 winter season, and closer to the threatening situation, in the pandemic of 1968. Furthermore, many countries do not have their own production facilities, and will rely on those countries that do. Will those countries be able to share vaccine supplies?

Osterholm asks (Osterholm 2005), "What if the pandemic were to start …"

• tonight
• within a year
• in ten years?

The New England Journal of Medicine had an interview with Dr Osterholm, which is available online for listening to or for downloading:

http://content.nejm.org/cgi/content/full/352/18/1839/DC1

If the pandemic were to start now, we would have to rely on non-vaccine measures for at least the first 6 months of the pandemic, and even then, the volumes produced would not be sufficient for everyone, and some sort of rationing or triage system would be necessary. Vaccine and drug production would have to be escalated - for much later in the pandemic, as this will not make a difference in the short term. The world's healthcare system would have to plan well in order to cope with distribution when they become available - at present, it is doubted that it could handle the distribution and administration of the vaccines, never mind trying to handle that under the pressure placed on it by a pandemic. Vaccines may only be available for the second wave of the pandemic, which tends to have a higher mortality than the initial wave.

If the pandemic starts in a year's time, it is likely that we will then have some experience in developing mock vaccines, so that a vaccine could be produced relatively quickly using a variety of the technologies currently under investigation. There would still be a significant delay, and it is likely that there would still be insufficient quantities, with rationing required.

We don't know when a pandemic will occur - but starting preparation now is essential. If the pandemic is delayed by a few years, we may well have the required vaccine production capacity to minimise the disastrous consequences.

The WHO suggests various strategies to solve these problems (WHO 2005d) and is working with governments, scientists, vaccine and drug companies, and other role players around the world to achieve a solution.

Shorten the time between emergence of a pandemic virus and the start of commercial production.

1. Candidate "pandemic-like" vaccines need to be made and put through trials. This will require adopting a centralized evaluation team to examine the findings of the studies and give clearance for the use of the vaccine. It would not be feasible for each medicine's evaluation team to do this for their own country. The vaccine needs to become established through "mock" trials in order to be able to be expedited in this way - then, like the current influenza vaccine, it is known, and only brief studies are required to confirm immunogenicity and safety.
2. Increased production capacity must be developed worldwide - for example, changing to cell culture vaccines. Another important means to improve production is to increase consumption - using more of the current vaccine today will not only decrease the burden of current influenza disease, as well as helping to prevent reassortment in humans infected with two strains of virus, but will ultimately enable production to be increased.

A number of controversies surrounding the development of a new influenza vaccine need to be dealt with (Fedson 2005, Osterholm 2005).

Financial - patents exist for the plasmid-based methods of making virus in cell culture and the legal implications in various countries need to be examined and addressed. Will the owners of the intellectual property benefit in any way? Mock vaccines need to be made, but will probably never be sold and used. Who will fund this endeavour?

Rationing - in the event of vaccine shortage, higher risk groups will need vaccination first, along with those working on the front lines to control the pandemic. In such an event, the definition of "high risk group" may need to be revised - will it include children, for instance? Who will get the vaccine first - there is already tension over this issue in the UK: poultry farmers or healthcare workers? (Day 2005)

Equitable access will need to be ensured - countries without vaccine production, poorer countries, and developing countries will all want to have their share of the vaccine supply.

Liability issues - due to increased vaccination with current vaccines, greater attention must be paid to liability. Several countries have legislation that limits and/or covers certain liability for vaccine companies - encouraging such legislation will make vaccine companies feel more free to develop new vaccines, and increase the supply of current vaccines. When the time comes for rapid entry of pandemic vaccines into general use, such legislation will be important.

Barnett employs a Haddon Matrix to show what sort of planning needs to be done at different stages of the pandemic, from pre-pandemic to post-pandemic (Barnett 2005).

The WHO will play an important role in the process. In 2001, the Global Agenda for Influenza Surveillance and Control was established (Webby 2003, Stohr 2005). Its role is to enhance our surveillance abilities, in order to better detect a pandemic, and prepare for influenza seasons until then. It is also charged with the task of increasing our knowledge of influenza, and enhancing vaccine acceptance and use, in order to prepare us for a pandemic (WHO 2005j).

The WHO also needs to lead the address of the problems of production capacity, legislation and expedited vaccine availability, and research that needs to be done in order to reach the point where these are possible. It needs to help solve the controversies over financing, patents and intellectual property, equity for developing countries and countries not producing vaccine, and rationing of vaccine when supplies do not meet the demands of a population of more than 6 billion people.

"Our goal should be to develop a new cell culture-based vaccine that includes antigens that are present in all subtypes of influenza virus, that do not change from year to year, and that can be made available to the entire world population. We need an international approach to public funding that will pay for the excess production capacity required during a pandemic." (Osterholm 2005)