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chosen to be represented in the vaccine is the same as that used in the previous vaccine, the process is faster.

First, the CDC, or other reference source, take the strains to be used and grow them in combination with a strain called PR8 (H1N1 A/PR/8/34) which is attenuated so that it is apathogenic and unable to replicate in humans (Beare 1975, Neumann 2005). This allows reassortment to occur, resulting in a virus containing six PR8 genes along with the haemagglutanin (HA) and neuraminidase (NA) of the seasonal strain. This new virus is then incubated in embryonated hens’ eggs for 2-3 days, after which the allantoic fluid is harvested, and the virus particles are centrifuged in a solution of increasing density to concentrate and purify them at a specific density. Then, the viruses are inactivated using formaldehyde or β-propiolactone, disrupted with detergent, and the HA and NA are purified. Finally, the concentrations are standardized by the amount of hemagglutination that occurs (Hilleman 2002, Potter 2004, Treanor 2004).

In about June/July, the strains are tested to ensure adequate yield, purity, and potency. After this, the three strains – two influenza A strains and one influenza B strain, which were all produced separately – are combined into one vaccine, their content verified, and packaged into syringes for distribution.

Production capacity

At present, the world has a production capacity of about 300 million trivalent influenza vaccines per year, most of which is produced in nine countries – Australia, Canada, France, Germany, Italy, Japan, the Netherlands, the United Kingdom, and the United States. In 2003, only 79 million doses were used outside of these countries and Western Europe. A further 13.8 million vaccines were produced and used locally in Hungary, Romania, and Russia (Fedson 2005).

Approximately 4-5 million doses of the live attenuated virus vaccine are produced per year.

Types of Influenza Vaccine

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 vaccines

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

Whole virus vaccines were the first to be developed. The influenza virus was grown in the allantoic sac of embryonated hens’ eggs, subsequently purified and concentrated using red blood cells, and finally, inactivated using formaldehyde or β- propiolactone. Later, this method of purification and concentration was replaced with centrifuge purification, and then by density gradient centrifugation, where virus particles of a specific density precipitate at a certain level in a solution of increasing density. Subsequently, filter-membrane purification was added to the methods available for purification/concentration (Hilleman 2002, Potter 2004).

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

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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.

Live vaccines

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).

Vaccines and technology in development

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.

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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 of the virus 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).

Attenuation by deletion of the gene NS1 or decreasing the activity of NS1 is being investigated. NS1 produces a protein that inhibits the function of interferon alpha (IFNα). If a wild-type influenza virus infects a person, the NS1 protein antagonises IFNα, which has an antiviral effect. An infection with a NS1-deficient virus would quickly be overcome by the immune system, hopefully resulting in an immune response, but with no symptoms (Palese 2002b).

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.