Polypeptides and DNA encoding same

Details Polypeptides and DNA encoding same Polypeptides and DNA encoding same

1999-05-24

2002-12-10

Guzo, David (Department: 1636)

Drug, bio-affecting and body treating compositions

Antigen, epitope, or other immunospecific immunoeffector

Amino acid sequence disclosed in whole or in part; or...

C424S184100, C424S191100, C424S265100, C424S268100, C424S269100, C424S272100, C514S002600, C514S04400A, C536S023100, C536S023700, C536S024100, C435S320100, C435S455000, C435S456000, C435S325000, C435S348000, C435S069100, C435S091400, C435S091410, C435S091420

Reexamination Certificate

active

06491920

ABSTRACT:

This invention relates to polypeptides, and to DNA encoding same, produced by human malaria parasites. It also relates to methods of preparing the polypeptides, to antibodies thereto and compositions for use against malaria.
Plasmodium falciparum
malaria is one of the most common infectious diseases in the world today, threatening up to 40% of the world's population. It is a disease of the Third World. There are between 150 and 300 million cases of this disease annually, over 1% of cases are fatal, babies and young children being the most vulnerable. With the advent of insecticides and new parasiticidal drugs developed after World War II it was felt that the disease could be eradicated. The early attempts proved very successful but with time the parasite has developed resistance to drugs such as chloroquine and the mosquito vector (Anopheles) has developed resistance to DDT. As a consequence of this it is necessary to develop new approaches to try to combat the disease. As immunity to the disease develops with increasing age, in endemic areas, a vaccine, together with new anti-malarials and insecticides need to be developed if the disease is to be eradicated.
Current research programmes, throughout the world, are involved in defining what antigens might form part of a useful vaccine. The complex life-cycle of the parasite means that a simple vaccine based on one antigen may not be adequate and that an effective vaccine will probably require antigens from different development stages.
The human malaria parasite,
Plasmodium falciparum
, has a complex life-cycle, during which different antigens are produced at particular developmental stages. The major antigen on the sporozoite surface is the circumsporozoite or CS protein, which probably determines the specificity of the interaction between the parasite and liver cells. CS protein contains two conserved amino acid sequences, known as regions I and II, which are separated by a repeating amino acid motif.
The cloning of the gene for this protein has permitted the development of various vaccines. To date vaccine trials using parts of the CS protein have proved disappointing. Immunity to sporozoites does not necessarily prevent the erythrocytic phase of the life-cycle which is associated with clinical disease. Only one sporozoite needs to evade the immune system for clinical disease to occur. Currently CS protein is the only well-characterised protein known to be involved in host-cell recognition. The merozoite is the developmental stage capable of re-infecting fresh red cells. Antibodies which prevent gametocyte differentiation within the mosquito are useful in breaking the transmission cycle as well. Another complexity is the antigenic variation displayed by the parasite. A vaccine against the asexual erythrocytic parasite, need only be partially effective to reduce the severity of the disease. A vaccine against the asexual blood stages of
P. falciparum
has been developed by Patarroyo et al (Nature Vol. 332, 1988, p158) based on the use of synthetic peptides, but this has not proved to be totally effective.
We have now found that polypeptides sharing certain sequence motifs with CS protein are produced during the erythrocytic or merozoite stage of the parasite life-cycle.
Accordingly, the present invention provides a polypeptide from the group comprising:
a) a polypeptide having the amino acid sequence of Formula I;
b) polypeptides having substantially the same structure and biological activity as a);
c) fragments, derivatives and mutants of a) or b) significantly involved in their biological activity;
d) oligomeric forms of a), b) or c) significantly involved in their biological activity.
It will be understood by those skilled in the art that some variation in structure may occur in naturally occurring biologically active polypeptides and that malarial proteins in particular display antigenic variability. Provided that structural variations do not eliminate the biological activity of interest such as, for example, involvement in parasite recognition of red cells, red cell attachment or merozoite invasion, the present invention includes such variations within its scope.
Thus, although formula I relates to a cloned isolate of
P. falciparum
from Thailand known as T.9/96, the scope of the invention also includes, for example, a polypeptide derived from another Thailand isolate known as K1. This was known to differ from T9/96 in lacking a Hinf 1 restriction site and having an extra Bgl II site, which has been confirmed by sequencing. Polypeptide from K1 differs from T9/96 in certain details as set out in Table 1 but the conserved regions are intact.
Both T9/96 and K1 are obtainable from the WHO Registry of Standard Strains of Malaria Parasite, Dept. of Genetics, University of Edinburgh, United Kingdom.
TABLE 1
Comparison of DNA and Polypeptide from T9/96 and K1
Nucleotide
Amino acid
Position in
Amino Acid
residue
codon
T9/96
K1
T9/96
K1
39
1
A
C
S
R
46
1
C
G
Q
E
83
3
T
A
D
E
90
1
G
C
V
L
92
1
G
A
V
I
98
2
A
G
K
R
119
2
G
A
R
K
134
2
C
G
T
S
179
2
G
A
S
N
277
1
A
T
I
L
290
1
T
C
W
R
297
1
G
C
D
H
311
2
C
T
S
F
312
1
T
G
S
A
314
1
C
G
Q
E
337
2
A
G
D
G
341
3
C
A
N
K
359
1
G
C
E
Q
361
1
A
G
K
E
398
2
A
T
H
L
412
   1, 2
AA
TC
N
S
490
2
A
G
E
G
Generally, about 5% variation in amino acid residues may be tolerated but, as will be understood by those skilled in the art, some regions of the molecule and some residues are more significant than others. Conserved regions which play an important role in biological activity are likely to be less tolerant of variation (e.g. in and around the region displayed for TRAP in Formula III), whereas antigenically important regions, for example around the RGD sequence (residues 307-309 of Formula I) are more subject to variability. TRAP as used herein is an abbreviation for “Thrombospondin related anonymous protein” and indicates one or more of the polypeptides of the present invention. Other regions may be somewhat less significant but there is some evidence of biological activity associated with NP or PN sequences. By “conserved” we mean having significant homology of amino acid residue sequences with other proteins of interest. Thus, for example, the region from about residue 244 to about residue 291 has significant homology with CS proteins from various strains of malaria parasite and with thombospondin and properdin framework proteins as illustrated in Formula III. It is not possible to put precise numerical limits on the degree of homology but 80% or greater say, would in many examples be expected to be significant.
The present invention also provides fragments of the above polypeptides, preferably containing a conserved sequence, for example, a fragment from the region extending from amino acid residues 244 to 291 of Formula I and more particularly a polypeptide selected from the following group;
a) WDEWSPCSVTCGKGTRSRKR
b) WDEWSPCSVTCGKGTR
c) EWSPCSVTCGKG
d) PCSVTCGKG
e) WSPCSVTCG
The single letters in the formula represent the following naturally occurring L-amino acids: (A) alanine, (C) cysteine, (D) aspartic acid, (E) glutamic acid, (F) phenylalanine, (G) glycine, (H) histidine, (I) isoleucine, (K) lysine, (L) leucine, (M) methionine, (N) asparagine, (P) proline, (Q) glutamine, (R) arginine, (S) serine, (T) threonine, (V) valine, (W) tryptophan, (Y) tyrosine.
Derivatives of the polypeptide of the invention are, for example, where functional groups, such as amino, hydroxyl, mercapto or carboxyl groups, are derivatised, e.g. glycosylated, acylated, amidated or esterified, respectively. In glycosylated derivatives an oligosaccharide is usually linked to asparagine, serine, threonine and/or lysine. Acylated derivatives are especially acylated by a naturally occurring organic or inorganic acid, e.g. acetic acid, phosphoric acid or sulphuric acid, which usually takes place at the N-terminal amino group, or at hydroxy groups, especially of tyrosine or serine, respectively. Esters are those of naturally occurring alcohols, e.g. methanol or ethanol.
Further derivatives are salts, e

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