Process for isolating the immunogenic components of influenza viruses

#The present invention relates to influenza vaccines, in particular influenza sub-unit vaccines, and their production by selective solubilisation and isolation of the immunogenic components of influenza virus.

#FIG. 1 is a schematic representation of the influenza virus particle. The genetic material, ribonucleic acid (RNA), associated with the group-specific nucleoprotein is surrounded by a double membrane consisting of an inner layer of protein and an outer layer of host-derived lipid material. Two glycoproteins, hemagglutinin and neuraminidase, appear as projections or spikes on the surface of the viral envelope.

#It is now well-established that the two glycoproteins, hemagglutinin and neuraminidase, are the major immunogenic components of the influenza virus, all other components, including other virus proteins, nucleic acid and lipids, being non-essential for the induction of immunity. However, the presence of such non-essential materials in an influenza vaccine may lead to undesired side effects and, in any effect, limits the dosage of the vaccine which can be administered and, consequently, the level of immunity which can be achieved.

#Influenza virus of the antigen type X-31 (recombination of the strain A.sub.2 /Aichi/68) is multiplied in embryonated chicken eggs by incubation at 37.degree. C for two days. The eggs are then chilled at 4.degree. C overnight and the harvested infected allantoic fluid pooled. The virus is subsequently concentrated and purified from the infected allantoic liquid by centrifuging in a continuous flow zonal centrifuge (model RK, Electro-Nucleonics) using a sucrose gradient in phosphate buffered saline. The virus concentrate obtained after reduction of the sucrose content to less than 5% by dialysis against phosphate buffered saline in the cold, has a hemagglutination titre of 1:2.sup.17 and a protein content of 0.7 mg/cc. The immunogens are split off by adding to the virus suspension 1/50 of its volume of an aqueous detergent solution (cetyltrimethylammonium bromide, 1% solution). After 30 to 60 minutes (room temperature) the reaction mixture is worked up by zonal gradient centrifuging using a preformed linear sucrose gradient and subsequent fractionation of the gradients with a peristaltic pump. Hemagglutinin and neuraminidase are solubilized quantitatively and are present in the upper part of the gradient, well separated from the virus residual particle which forms a sediment much more rapidly.

#Multiplication, concentration and cleavage of the virus are effected as described in Example 1. Working up is effected by equilibrium centrifuging in a preformed sucrose gradient. After adjusting equilibrium, the gradient is fractionated and tested: hemagglutinin and neuraminidase are present in the lighter part of the gradient, well separated from the more dense virus residual particle.

#The process is effected as described in Example 1 or 2, except that influenza strain MRC-2 (recombination of type A.sub.2 /England/42/72) or MRC-11 (recombination of type A.sub.2 /Port Chalmers/73) is used.