Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector
Reexamination Certificate
1997-07-31
2001-01-16
Minnifield, Nita (Department: 1645)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
C424S188100, C424S186100, C530S350000, C530S324000, C530S300000, C530S333000, C514S002600
Reexamination Certificate
active
06174528
ABSTRACT:
The present invention relates generally to chimeric peptides comprising one or more protective epitopes in a conformation enabling immunological interactivity and to vaccine compositions comprising same. The present invention is particularly directed to a chimeric peptide capable of inducing protective antibodies against Group A streptococci.
Bibliographic details of the publications referred to in this specification by author are collected at the end of the description. Sequence Identity Numbers (SEQ ID NOs.) for the amino acid sequences referred to in the specification are defined following the bibliography.
Throughout this specification, unless the context requires otherwise, the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.
Many proteins which may be useful vaccine candidates against several diseases have a coiled-coil structure, an important structural and biologically abundant motif found in a diverse group of proteins (Cohen and Parry, 1990, 1986). More than 200 proteins have now been predicted to contain coiled coil domains (Lupas et al., 1991). These include surface proteins of certain bacteria such as streptococcal protein A and M proteins; viruses such as influenza hemagglutinin and human immunodeficiency virus (HIV) glycoprotein gp45; and protozoa such as VSG of Trypanosomes. All coiled coil motifs share a characteristic seven amino acid residue repeat (a-b-c-d-e-f-g)
n
. The x-ray structure of several coiled-coil domains have been solved and these include the leucine zipper portion of the yeast transcription factor GCN4 dimer (O'Shea et al. 1991), the repeat motif of &agr;-spectrin (Yan, 1993), together with the GCN4 leucine zipper trimer (Harbury et al., 1994) and tetramer (Harbury et al., 1993) mutants.
In the development of a subunit vaccine based on these proteins, it is generally difficult to map epitopes within the coiled coil structure. Furthermore, protective epitopes may need to be presented in the correct conformation for immunological recognition, such as antibody binding. This is especially important in defining a stable minimal epitope and using it as a vaccine.
Group A streptococci (hereinafter referred to as “GAS”) are the causative agent of several human diseases and can lead to acute rheumatic fever which causes serious heart disease. Rheumatic fever may represent an autoimmune illness initiated by cross-interactivity between the streptococcal M protein and cardiac antigens (Beachey et al., 1988). The M protein contains a seven-residue periodicity strongly suggesting that the central rod region of the molecule is in a coiled-coil conformation (Manula and Fischetti, 1980). Overlapping peptides have been made that span this region (see International Patent Application No. PCT/AU93/00131 [WO 93/21220]) and mouse antibodies raised against one synthetic 20mer peptide (designated “p145”) from the highly conserved C-terminal region can opsonise and kill multiple isolates of GAS (Pruksakorn et al., 1994a). In addition, p145 can inhibit in vitro killing mediated by human sera. Of concern is that p145 may also stimulate heart cross-reactive T cells (Pruksakorn et al., 1992; 1994b). The B cell epitope within p145 is thought to be conformational because truncated peptides fail to elicit a protective antibody response (Pruksakorn, 1994). There is a need, therefore, to define the minimal region of p145 that is required to induce opsonic antibodies; this could then form the basis of a vaccine. Such a method would enable the identification of the minimal epitopic regions from a range of proteins from pathogens.
One method that has been used to map minimal epitopes from antigens is the PEPSCAN method (Geysen et al., 1987). However, the short peptides used only indicate sequential or continuous epitopes. Other methods to determine conformational epitopes, that is epitopes formed by the tertiary structure of the protein, rely upon mimotope strategies. A mimotope is a mimic of the epitope which induces the antibody. Peptides can be synthesised on polypropylene pins covering the total repertoire of octapeptides which can be made using the 20 common amino acids, i.e. 20
8
peptides (Geysen et al., 1987). Alternatively, an epitope library consisting of a vast mixture of filamentous phage clones, each displaying one peptide sequence on the virion surface, can be surveyed for antibody recognition (Scott and Smith, 1990).
In accordance with the present invention, overlapping peptides derived from a conformational epitope are embedded within a peptide having a similar native conformation. This approach has the potential to be used in the mapping of a range of conformational epitopes and design of minimal epitopes as vaccine candidates against GAS and a variety of other pathogens.
Accordingly, one aspect of the present invention contemplates a chimeric peptide comprising a first amino acid sequence comprising a conformational epitope inserted within a second amino acid sequence wherein said first and second amino acid sequences are derived from peptides, polypeptides or proteins having similar native conformations.
In accordance with this aspect of the present invention, the second amino acid sequence constitutes a “framework peptide” and provides an appropriate conformation for the chimeric peptide. A framework peptide is selected or otherwise engineered to provide a similar conformation to the first amino acid sequence such as in its naturally occurring form. In its most preferred embodiment, the framework peptide assumes a &agr;-helical coiled coil conformation and is, therefore, useful in presenting epitopes present in the first amino acid sequence in a similar conformation, i.e. an &agr;-helical coiled coil conformation.
According to this preferred aspect of the present invention there is provided a chimeric peptide comprising a first amino acid sequence comprising a conformational epitope inserted within a second amino acid sequence wherein said second amino acid sequence folds to an &agr;-helical coiled coil conformation.
The present invention is particularly exemplified herein by the first amino acid sequence being derived from the streptococcal M protein and in particular comprising a B-cell conformational epitope from within the following amino acid sequence (using the single letter abbreviation for amino acid residues):
L R R D L D A S R E A K K Q V E K A L E (SEQ ID NO:1),
or functional and/or chemical equivalents of one or more of these amino acid residues.
Accordingly, a particularly preferred embodiment of the present invention is directed to a chimeric peptide comprising a first amino acid sequence having at least three amino acids selected from within the following sequence:
L R R D L D A S R E A K K Q V E K A L E (SEQ ID NO:1),
wherein said at least three amino acids constitute a conformational B-cell epitope from streptococcal M protein and wherein said first amino acid sequence is inserted within a second amino acid sequence capable of folding to an &agr;-helical coiled coil conformation. Preferably, the first amino acid sequence comprises at least five, more preferably at least ten and even more preferably at least fifteen contiguous amino acid residues.
Alternatively, non-contiguous amino acids may be selected such as those on the outside face of the helix and which are required or sufficient for activity.
The construction of a framework peptide is based on the seven amino acid residue repeat:
(a-b-c-d-e-f-g)
n
where a and d positions preferably have large apolar residues, positions b, c and f are generally polar and charged and positions e and g generally favour interchain ionic interactions. A particularly preferred framework peptide is based on the structure of a peptide corresponding to GCN4 leucine zipper (O'Shea et al., 1989; 1991) or its trimer (Harbury et al., 1994) or tetramer (Harbury et al., 1993) and the rep
Cooper Juan Anton
Good Michael Francis
Relf Wendy Anne
Saul Allan James
Counsel of the Queensland Institute of Medical Research
Fulbright & Jaworski
Minnifield Nita
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