Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Fusion protein or fusion polypeptide
Patent
1993-11-08
1996-07-09
Sidberry, Hazel F.
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Fusion protein or fusion polypeptide
4241841, 4242341, 4242041, 4242011, 4242031, 435 693, 4351723, 4352351, 4353201, 514 2, 536 231, 536 2372, 530350, 530825, 530826, A61K 39116, A61K 3912, C07K 14195, C12N 701
Patent
active
055342574
DESCRIPTION:
BRIEF SUMMARY
This invention relates to an antigen-presenting chimaeric protein and is particularly directed to virus proteins containing foreign epitopes, the preparation of such proteins and their use as vaccines.
The growth of recombinant DNA technology in recent years has led to the introduction of vaccines in which an immunogenic protein has been identified, cloned and expressed in a suitable bacterial host to obtain sufficient quantities of protein to allow effective protective immunisation in both animals and humans. As an extension of such techniques, it has been proposed to incorporate only the immunogenic epitopes in carrier proteins capable of expression in a suitable host while retaining the immunising properties of the epitope. Thus Beesley et al., Biotechnology, 8, 1990, 644-649, describe the preparation of chimaeric proteins in which epitopes of foot and mouth disease virus or human chorionic gonadotrophin are fused to the N-terminus of hepatitis B core protein as carrier. Expression of the chimaeric protein was carried out in yeast, as expression in E. coli was found to be not wholly satisfactory.
It has also been suggested by Pushko et al., Abstracts of VIIIth International Congress of Virology, Berlin, Aug. 26-31 1990, P38-006, that certain unidentified regions of the RNA-phage fr coat protein capsid may be capable of accepting foreign amino acid sequences without loss of capsid-forming ability. However, these workers give no indication of anything other than randon insertion of such amino acid sequences throughout the coat protein so as to obtain certain unidentified chimaeric constructs.
There is clearly a need in the furtherance of vaccine technology for production of chimaeric proteins capable of efficient and reproducible presentation of foreign immunogenic epitopes, which proteins can be made by recombinant methods involving expression in a well understood and regulatable bacterial host such as E. coli. Natural viral epitopes are of course presented on the regular surface lattice of viral shells or membranes. A system which mimics this natural form of antigen presentation should, in theory, prove particularly efficient in eliciting the immune response. However, to achieve such a result, it is necessary to identify bacterial virus coat proteins which can be cloned and when expressed form capsids independently of the genetic material of the virus. Furthermore, it is necessary to identify a particular region of the coat protein which is not essential to capsid formation and which can be manipulated independently of the remainder of the protein so as to receive the foreign epitope.
It has now been found that eptiope insertion in an identified class of virus protein carriers can be specifically directed so that foreign epitopes are reliably presented on the surface of the protein capsid assembly after the expression of the chimaeric protein in a bacterial host.
It was by no means predictable that such a result could be achieved. Not all viruses possess coat proteins which will self assemble: furthermore it is not possible to predict such self assembly. Even further, unlike animal viruses, bacterial viruses cannot naturally be expected to possess immunodominant regions and there is thus no guidance as to which region of a bacterial virus coat protein (if any) could be modified in the reasonable expectation of inducing an immune response.
According to the present invention there is provided a chimaeric protein, capable of forming part of a capsid assembly and comprising the amino acid sequence of the coat protein of phage MS-2, or a conservatively modified variant thereof, or sufficient of said sequence or variant to retain capsid-forming capability, which amino acid sequence has been modified by insertion of a foreign eptiope in the region of the protein corresponding to an N-terminal .beta.-hairpin, as determined by X-ray crystallography of the whole phage particle.
Surprisingly, it has been found that such a chimaeric protein can be expressed in a suitable bacterial host to yield capsids empty of the
REFERENCES:
Bowie, J. V. et al. Science 247: 1306-1310 (1990).
Kumar, V. et al. Proc. Natl. Acad. Sci USA 87: 1337-1341 (1990).
de la Cruz et al. J. Biol. Chem. 263 (9): 4318-4322 (1988).
Smith, G. P. Science 228: 1315-1317 (1985).
Peabody, D. S. J. Biol. Chem. 265(10): 5684-5689 (1990).
Kozcovskaya, T. M. et al. J. Uol. Biol (USSR) 584-593 (1988).
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Reisnier et al, Thrombosis and Haemostasis, 58, No. 1 (1987) 353, Abstract.
Mastico Robert A.
Stockley Peter G.
Talbot Simon J.
British Technology Group Limited
Sidberry Hazel F.
Tuscan Michael
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