The Evolving History of Influenza Viruses and Influenza Vaccines

Claude Hannoun


Expert Rev Vaccines. 2013;12(9):1085-1094. 

In This Article

Virus Production & Purification

The highest yields of influenza virus in allantoic fluid were obtained with low multiplicity of infection (ratio of infectious virus particles to cells), and strains that were highly infectious but caused a delayed apoptosis of the infected cells. These criteria are taken into account in the selection of vaccine strains and the production processes.[12] The PR8 strain, that had undergone a large number of passages in various hosts, corresponded well to these criteria. Unlike these well-adapted strains, such as PR8, the more recently isolated strains have undergone fewer passages in chick embryos and have low replication rates making them not so well adapted for mass production. However, our improved knowledge about influenza virus structure and its biological properties have enabled us to use genetic reassortment techniques to modify 'wild-type' strains so as to increase their yield in culture.

The genome of the influenza virus is divided into eight segments of RNA, each responsible for coding for different proteins. Some of these proteins are located on the virus surface such as hemagglutinin (HA) and neuraminidase (NA) and these play an important role in immunology and epidemiology of influenza. Other internal proteins, such as nucleoprotein, are involved in viral replication. Co-infection of cells with a wild-type strain and a strain that can replicate rapidly leads to exchange of RNA segments so that the resulting 'reassorted' strains have the antigenic properties of the wild-type (surface proteins) and replication properties of the culture-adapted strain (internal proteins). For influenza A, the PR8 strain is often used as a donor for this latter property. The reassortant viruses have two important proprieties: rapid replication and late induction of apoptosis following intracellular replication. In addition, they have the necessary antigenic characteristics to respond to the anticipated epidemiological situation. More recently, using reverse genetic methods and then genetically modified organisms, it has become possible to insert a specific gene into the viral genome with more precision than with reassortant techniques thereby enabling a more controlled modification of the selected protein.[13] Reverse genetic methods involve transfecting plasmid DNA, which encodes the influenza antigens, into mammalian cells for the production of attenuated viruses. Six plasmids containing genes for internal protein from an approved, high-growing master strain are transfected along with two plasmids containing the HA and NA genes from the highly pathogenic, wild-type target strain. This produces a 6:2 recombinant virus, which contains HA and NA segments from the wild-type virus and the remaining six segments from the high-growing strain, without the highly pathogenic properties. It has been shown that the 6:2 recombinant virus is able to grow well in eggs.

Until recently, all influenza vaccines were prepared by culturing the virus in embryonated hen eggs. Since 2007, new vaccines containing cell-cultured virus have become available; some of these vaccines have been licensed by the european medicines agency (EMA) and so can be used in EU countries. This method avoids the use of hen eggs, which are sometimes difficult to obtain in large quantities, especially if there is an epizootic outbreak in poultry. Allergic side reactions can be expected to be less dangerous, scaling-up and production automatization would be simpler and sterility problems easier to solve. Only some continuous cell culture lines such as Vero (monkey) or MDCK (dog) cells have been successfully used after establishment of strictly controlled cell banks. The technical developments are not simple and currently, only a small proportion of the available influenza vaccines are produced in cell culture.

The virus that is grown in embryonated hen eggs is harvested as a relatively clear liquid (allantoic fluid) containing few contaminants. These contaminants are fairly easy to remove using the purification process which has evolved with the standards for vaccine purity that aim to reduce possible adverse effects due to the presence of non-viral components. The first purification technique, which involved the adsorption of the virus onto chick red blood cells at 4°C and then elution at 37°C, was specific but yielded only partially purified virus.[14]

A more technical method involving differential centrifugation of the allantoic fluid at a high speed (60,000 rpm) to separate the virus from soluble contaminants by sedimentation was developed.[15] After removal of the supernatant, the virus, which is in the pellet, can be resuspended in appropriate diluent solution. This approach has since been improved using a technique known as density gradient or differential centrifugation in which the virus is centrifuged at high speed in a buffered sucrose gradient. During centrifugation, the virus forms a band at its corresponding density in the gradient, making it easy to harvest in a highly purified form. An improvement of this process, known as continuous flow centrifugation, using complex continuous flow machines similar to industrial cream separators, enables large quantities of allantoic fluid to be processed enabling the process to be scaled up to industrial conditions.