L2 immunogenic peptides of papillomavirus

Details L2 immunogenic peptides of papillomavirus L2 immunogenic peptides of papillomavirus

1997-05-12

2001-01-16

Salimi, Ali (Department: 1645)

Drug, bio-affecting and body treating compositions

Antigen, epitope, or other immunospecific immunoeffector

Virus or component thereof

C424S186100, C424S192100, C435S069100, C435S069300, C530S300000, C536S023720, C536S023400

Reexamination Certificate

active

06174532

ABSTRACT:

RELATED APPLICATIONS
This application claims priority to International Application No. PCT/GB95/02372, filed Oct. 6, 1994, which is based on British Application No. GB 9420146.4, filed Oct. 6, 1994.
FIELD OF THE INVENTION
The present invention relates to the use of papillomavirus L2 protein fragments in medicine. In particular the invention relates to vaccine preparations for immunisation against papillomavirus and papillomavirus related diseases in mammals, particularly humans and cattle.
BACKGROUND OF THE INVENTION
Papillomaviruses induce a variety of lesions both in humans and in animals. Some papillomas, albeit benign, are themselves a clinical problem, such as laryngeal papillomas of children (Steinberg and Abramson, 1985) or penile papillomas of bulls (Jarrett, 1985a), and others are known to be a risk factor in the pathogenesis of cancer, as in the case of flat lesions of the cervix or penile condylomata in humans (zur Hausen, 1978). Therefore both in human and veterinary medicine the introduction of an anti viral vaccine, would be of major importance.
International Patent Application No. PCT/GB 92/01092 relates to the use of papillomavirus L2 protein as a therapeutically effective component in the treatment of papillomavirus tumours or lesions. In the examples, it is stated that the L2 open reading frame (ORF) of BPV-4 was cloned as the whole L2 ORF (encoding amino acids—8 to 525) and as three fragments encoding amino acids 11 to 201, 203 to 329 and 330 to 525. In subsequent vaccination experiments mixtures of the above amino acid sequence fragments were used in vaccination compositions. However, the identification of the active fraction capable of conferring immunity to papillomavirus and by extension to papillomavirus related diseases was not attempted. Other workers have reported that the L2 protein of cottontail rabbit papillomavirus (CRPV) is a prophylactic vaccine in rabbits [Christensen et al., Virology, (181, pp 572-579 (1991); Lin et al., Virology, 187, pp 612-619 (1992)] and the L2 protein of BPV-4 in cows (Campo et al, J.gen.virol. 74, pp 945-953 (1993). Christensen et al used the terminal carboxyl half of the L2 protein (amino acids 259-492) of CRPV and Lin et al used an L2 protein of CRPV from amino acid number 67-489. Neither group suggested employing a polypeptide comprising a portion corresponding to the active N-terminus of BPV-4 L2 (amino acids 11-200). Further workers, expressed the complete BPV-1 L1 and L2 proteins in
E. Coli
. The L1 protein was used to vaccinate calves and this resulted in a degree of protection from challenge with BPV-1 (Pilacinski et al., Papillomaviruses: Molecular and Clinical Aspects, UCLA Symposium 2, (1985), pp 257-271). It was also reported that rabbit antisera raised against L1 or L2 were capable of neutralising BPV-1 in transformation assays of C127 mouse cells, implying that both L1 and L2 contained neutralising epitopes. This was not tested in cows. Furthermore, only one in five rabbit L2 immune sera tested could neutralise the virus.
However, the prior art does not indicate which element of the papillomavirus L2 protein is the immunogenic element responsible for effective prophylactic therapy, though the work of Christensen et al. and Lin et al. would incline the skilled worker to expect the immunogenic epitope to lie within the carboxy-terminal portion of the peptide. Vaccines produced to date are generally made up of mixtures of various fusion proteins (if produced via recombinant DNA technology) or are of crude extracts of papillomavirus elements or are made up of live virus strains which may display an immunogenic activity in an animal species but which activity may or may not be readily reproducible. Such extracts used as vaccines have not generally been found to work well when applied to other species and do not appear to be particularly efficient.
It is an object of the present invention to provide a more effective prophylactic vaccine against papillomavirus infection in mammals.
It is a further object of the invention to provide a more efficient immunogenically active component for use in a prophylactic treatment of papillomavirus infection.
These and other objects will become apparent from the following description and examples.
SUMMARY OF THE INVENTION
It has now surprisingly been found that the immunogenic epitope or epitopes of the L2 protein lie within the N-terminal region, contrary to preliminary work (as described herein) and expectations of the present inventors and the inferences to be drawn from the teachings of the prior art. Thus, the N-terminal fragment of papillomavirus L2 (e.g. amino acids 11-200) or part thereof can help prevent the occurrence of disease states in mammals which are susceptible to papillomavirus infection.
Furthermore, it has been found that at least some of the epitopes found in the N-terminal region display a remarkable sequence similarity to the corresponding fragment of human papillomavirus types. Thus, the present invention provides the use of the N-terminal portion of papillomavirus L2 protein or a prophylactically effective peptide fragment thereof (or a prophylactically effective peptide derivative sequence thereof) in the production of a medicament suitable for use as a prophylactic agent against papillomavirus infection in mammals (which includes humans).
The peptide fragment can be upto about 200 amino acids long, preferably from about 10 to 200 amino acids long, more preferably from about 10 to 30 amino acids long, and most preferably about 10 to 20 amino acids long. In embodiments of the invention the immunogenically effective peptide fragment includes as a part thereof one of the following amino acid sequences (referred to as SEQ. ID NO:1, SEQ. ID NO:2 and SEQ. ID NO:3 reading from N-terminus to C-terminus and in particular the underlined amino acid fragment; or derivatives thereof so long as such derivatives of the peptide(s) has/have a prophylactic activity.
1  THR GLY VAL PRO ILE ASP PRO ALA VAL 
PRO
 10
SEQ ID NO:1

11 
ASP SER SER ILE VAL PRO LEU LEU GLU
 SER 20

1  GLY ALA GLU ILE GLU ILE ILE ALA GLU VAL 10
SEQ ID NO:2

11 HIS PRO PRO PRO VAL TYR GLU GLY PRO GLU 20

1  
VAL THR ILE GLY ASP ILE GLU GLU PRO PRO 10
SEQ ID NO:3

11 
ILE LEU GLU VAL
 VAL PRO GLU THR HIS PRO 20

21 THR
Without the intention of being bound by theory it is thought that the above amino acid sequences (and particularly the underlined fragments) may not be capable of eliciting an immune response per se, but are generally required to be included as a part of a larger protein if a satisfactory immune response is to be obtained. The amino acid sequence may thus be part of a native papillomavirus L2 sequence, or a synthetically derived like sequence thereto, or may be conjugated or fused to another protein or peptide such as keyhole limpet haemocyanin (KLH) (for rabbits) or glutathione-S-transferase (GST).
Thus, the invention also includes isolated or synthetically prepared peptides which are capable of prophylactic function and therefore possess an appropriate immunogenic capacity.
For the purposes of the present invention a derivative of a sequence of the invention is one which is capable of eliciting a prophylactic effect in a mammal. Preferably such a derivative retains a recognisable degree of similarity with respect to one of the L2 sequences shown hereinabove. Thus, substitutions, additions, deletions or combinations thereof can be made in the sequence of amino acid residues provided that any resultant sequence is capable of eliciting a prophylactic effect in an appropriate protein, polypeptide, or peptide format. Focusing on the ca 20 amino acid residue long BPV-L2 sequences (I) and (III) per se, the degree of similarity

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