Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1998-05-13
2002-04-30
Wortman, Donna C. (Department: 1648)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007100, C436S518000
Reexamination Certificate
active
06379905
ABSTRACT:
The present invention relates to synthetic polypeptides. In particular it relates to synthetic polypeptides which emulate the three-dimensional structures and/or electrostatic surfaces and/or other physical, chemical and structural properties of specific regions of proteins thought to be the involved in the molecular pathology of spongiform encephalopathies. It is of particular interest to the design of immunodiagnostics, vaccines and other medical, veterinary or scientific agents in relation to human, bovine and ovine spongiform encephalopathies.
Spongiform encephalopathies are a group of, degenerative neurological diseases. Examples have been found in a number of species including sheep (where it is known as scrapie), cows (BSE) and humans (Creutzfeldt-Jakob disease (CJD) and kuru) (Review article, Taylor, D. M. Veterinary Record 125,413-415 (1989)). Similar conditions have also been found in the wild mink population and in captive kudus (a kind of antelope) and tigers. It has been variously reported that BSE can be transmitted under laboratory conditions to mice and pigs. This crossing of species barriers by the infective agent has led to increased concern that transfer to humans could occur.
These diseases are characterised by a slow incubation time of four to five years after which the clinical symptoms of progressive degeneration of mental state, including aggressiveness and lack of coordination, appear. Post mortems reveal a characteristic pattern of vacuolation in brain tissue due to the destruction of neural cells, and the deposition of unusual protein fibres.
Although the form of the disease found in sheep (scrapie) has been known for many years, spongiform encephalopathies have come to prominence within the last decade following the appearance of BSE in cattle farms. The incidence of BSE in the United Kingdom has increased markedly during this period and public concern over the possible transmission of the disease to humans has led to a collapse in the beef market. Thus for both veterinary and economic reasons, there is an urgent need for diagnostic agents to detect infection and for vaccines to prevent infection.
It is believed that the causative agent of scrapie and its counterparts in other animals is a so-called “prion”, that is an infective particle comprising protein only and no nucleic acid, the presence of the latter being required in the case of a conventional virus. In scrapie, one particular protein (termed prion protein, PrP
sc
) has been found to co-purify with infectivity and can produce a scrapie-like condition in brain cell cultures from other animals, such as hamsters, under laboratory conditions. PrP
sc
is the only known component of the characteristic protein fibres deposited in the brain tissue of scrapie-infected sheep. The term “PrP
sc
” as used herein should be taken to refer not only to the specific Prion protein identified in sheep but also to those homologous proteins found in many other species which appear to undergo a structural modification as described hereinafter. The term “PrP
c
” shall be used in respect of the normal cellular counterpart to PrP
sc
.
The major problem in the search for a specific diagnostic agent or synthetic vaccine against the scrapie agent PrP
sc
is that it is almost identical to the natural form of the protein PrP
c
. The natural function of this protein. is not yet understood but the remarkably strong conservation of primary structure between homologous proteins from different species suggests that it has an essential structural or functional role within the organism.
In spite of the almost identical form of these prions to the natural proteins, we have deduced synthetic peptide structures comprising at least one antigenic property, such as an epitopic site and these synthetic peptides may be used to produce diagnostic agents and vaccines.
The responses of the B and T cells of the immune system are not specified by a global recognition of a whole protein but rather by recognition of a small region of the protein surface known as epitopic site. Such sites may be formed by a continuous section of peptide chain or may be discontinuous, where separated sections of peptide chain are brought together at the protein surface due to folding of the chain. One aim in producing a synthetic peptide vaccine is to mimic the structure of a particular epitope and thereby cause a primary immune response leading to the production of memory B cells which will secrete antibodies on subsequent exposure to the parent protein so producing a greatly enhanced response to secondary infection. A similar mechanism via priming of the cytotoxic T cells to respond more vigorously to a particular antigen will also occur.
However, problems exist with the application of traditional methods of vaccine production to this disease as it is believed that the molecular structure of the protein prion rather than nucleic acid sequence passes on infectivity in the prion. The usual method of viral vaccine production involves the inactivation of the virus in some way to destroy infectivity whilst preserving epitopic sites. Such techniques as heat treatment or serial passaging of the virus through a culture are used, but these approaches would not lead to a loss of infectivity of a prion unless conditions were such as to cause protein denaturation. If the conditions are severe enough to inactivate the prion protein then denaturation of the protein occurs and any epitopic sites are lost. Thus there is a major problem in trying to obtain antigenic but non-infective prion proteins by conventional routes. It is known, for example, that the scrapie agent in sheep is particularly resistant to chemical or physical inactivation (Hodgson,, J. Bio/Technology 8 990 (1990)).
In one aspect our invention provides a synthetic polypeptide having at least one antigenic site of a prion protein. Preferably the prion protein is of a form which only exists in nervous tissue of a mammal suffering from spongiform encephalopathy.
We have found that prion proteins of the type mentioned above comprise six regions of interest, labelled A to F, and two related frame shift peptide sequences, viz:1) a repeating section in region E having undergone a nucleic acid coding sequence frame shift of +1 (FSa) and 2) the repeating section in region E having undergone a nucleic acid coding sequence frame shift of −1 (FSb).
With regard to region A, our invention provides a synthetic peptide sequence according to general Formula (I): (SEQ ID NO: 52)
X-(R
1
-Lys-His-R
2
)-Ala-Gly-Ala-Ala-Ala-R
3
-Gly-Ala-Val-—Val-Gly-Gly-Leu-Gly-Gly-Tyr-Met-Leu-Gly-Ser-Ala-Met-Ser-(Arg-Pro-R
4
-R
5
)-Y (I)
wherein R
1
is an amino acid residue selected from Met, Leu and Phe;
R
2
is either Met or Val;
R
3
is Ala or is absent;
R
4
and R
5
are independently an amino acid residue selected from Leu, Ile and Met; one or more residues within brackets maybe present or absent with the proviso that if they are present they are attached to the rest of the peptide in sequence; and X and Y may each independently be absent or independently be one or more additional amino acid residues.
It will be apparent for example that the residues at the N-terminal of the sequence may be present as “R
2
”- or “His-R
2
-,” or “Lys-His-R
2
-” or “R
1
-Lys-His-R
2
-.” Similarly, the preferable residues at the C-terminal may be present as “-Arg”, or “-Arg-Pro,” or “-Arg-Pro-R
4
,” or “-Arg-Pro-R
4
-R
5
.”
Preferably, R
1
, if present, is Met, R
3
is Ala and R
5
, if present, is Ile. Also, if R
2
is Met then R
4
, if present, is Ile. Below are preferred sequences (Seq. I.D. No: 1 and Seq. I.D. No: 2) of formula I relating to bovine and ovine and to human prion proteins respectively:
Seq. I.D. No: 1
X-(Met-Lys-His-Val)-Ala-Gly-Ala-Ala-Ala-Ala-Gly-Ala-Val-Val-Gly-Gly-Leu-Gly-Gly-Tyr-Met-Leu-Gly-Ser-Ala-Met-Ser-(Arg-Pro-Leu-Ile)-Y; and
Seq. I.D. No: 2
X-(Met-Lys-His-Met)-Ala-Gly-Ala-Ala-Ala-Ala-Gly-Ala-Val-Val-Gly-Gly-Leu-Gly-Gly-Tyr-Met-Leu-Gly-Ser-Ala-Met-Ser-(Arg-Pro-Ile-Ile)-Y.
A particularly preferred sequence acco
Fishleigh Robert Vincent
Mee Roger Paul
Robson Barry
Pennie & Edmonds LLP
Proteus Molecular Design Limited
Wortman Donna C.
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