Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Virus or component thereof
Patent
1997-05-27
2000-04-11
Mosher, Mary E.
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Virus or component thereof
4242101, 4352351, 435326, 435329, A61K 35145, C12N 702, C12N 706
Patent
active
06048537&
DESCRIPTION:
BRIEF SUMMARY
The invention relates to a method for the fragmentation of live influenza virus, which is simple to carry out, leads to a destruction of the infectious power and can be used, in particular, in the manufacture of an inactivated influenza vaccine or for obtaining viral antigens for diagnostic purposes.
The influenza virus or flu virus is an enveloped, single-stranded RNA virus with helical symmetry, belonging to the family Orthomyxoviridae. The known antigens are essentially represented by the following structural proteins:
The nucleoprotein and the M protein are the internal antigens specific to the viral types A and B, the haemagglutinin and the neuraminidase determining the virus A subtypes.
The haemagglutinin is the most immunogenic glycoprotein of the viral constituents. The haemagglutinin content is used to express the load of viral antigens in influenza vaccines; nevertheless, it is now established that all of the viral antigens participate in the stimulation of the immune response, and that the internal antigens play a major part in the cell-mediated response.
The nucleoprotein (NP) is the main target antigen of cytotoxic T lymphocytes (CTL), no CTL specific for the haemagglutinin having been detected in man.
Viral fragmentation enables the envelope to be ruptured and all of the NP, M, RNA, viral polymerase, HA and NA viral antigenic sites to be released.
Vaccines obtained by this method are hence extremely immunogenic and give larger seroconversions than those obtained with vaccines containing only the surface antigens (which represent ony 36% of the viral constituents).
Vaccines containing fragmented virions are also better tolerated than vaccines containing whole virions, which are more allergenic.
The fragmentation methods used combine solvent-detergent treatments such as polysorbate/anaesthetic ether or polysorbate/chloroform [Pasteur Institute (Adamowicz-Muller) U.S. Pat. No. 4,522,809].
The implementation of these methods on an industrial scale necessitates suitable premises and equipment, such as flameproof installations in the case of ether and specific protection for the operators with respect to the neurotropic toxicity of chloroform.
Other methods employ detergents or proteolytic enzymes or bile salts, applied to previously inactivated viruses. These methods are essentially used for obtaining surface subunits (HA and NA) in the manufacture of subunit vaccines lacking the internal antigens. Examples which may be mentioned include the use of sodium deoxycholate described in Patent DD 155875, that of surfactants described in U.S. Pat. No. 4,327,182, and lastly that of nonionic detergents described in Patent FR 2,483,779.
Our method, which is simple to carry out, enables the fragmentation of purified live influenza virus to be performed without a solvent at room temperature. This method comprises the removal of the detergent and of undesirable substances, enabling the viral fragmentation product to be obtained in a fully defined, isotonic, buffered ionic environment.
This method leads to a very considerable reduction in the infectious titre of the purified influenza virus suspension, which can go as far as complete inactivation, depending on the working conditions adopted, such as the concentration and contact time between detergent and influenza virus.
Hence the subject of the present invention is a method for preparing purified influenza antigens from a fluid containing influenza virus, comprising the steps of concentration, purification, fragmentation and, where appropriate, inactivation, characterized in that: separated by a filtration step, of an amphiphilic nonionic detergent, followed by a removal of undesirable substances by filtration, retaining all of the viral constituents,
In a preferred embodiment of the method, the influenza virus used is obtained by culture on sensitive host cells, such as mammalian cells, namely monkey, hamster or pig kidney cells or ferret or mouse cells, or cells originating from embryos, from human pulmonary tissue or from chick embryo fibroblasts.
The comm
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Chalumeau Herve
Court Guy
Gerdil Catherine
McVerry Patric
Violay Jean Michel
Mosher Mary E.
Pasteur Merieux Serums et Vaccins
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