Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Amino acid sequence disclosed in whole or in part; or...
Reexamination Certificate
1998-03-30
2003-11-04
Swartz, Rodney P (Department: 1645)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Amino acid sequence disclosed in whole or in part; or...
C424S168100, C424S248100, C435S091100, C435S253100, C435S287200, C536S022100
Reexamination Certificate
active
06641814
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a number of immunologically active, novel polypeptide fragments derived from the
Mycobacterium tuberculosis,
vaccines and other immunologic compositions containing the fragments as immunogenic components, and methods of production and use of the polypeptides. The invention also relates to novel nucleic acid fragments derived from
M. tuberculosis
which are useful in the preparation of the polypeptide fragments of the invention or in the diagnosis of infection with
M. tuberculosis.
The invention further relates to certain fusion polypeptides, notably fusions between ESAT-6 and MPT59.
BACKGROUND OF THE INVENTION
Human tuberculosis (hereinafter designated “TB”) caused by
Mycobacterium tuberculosis
is a severe global health problem responsible for approximately 3 million deaths annually, according to the WHO. The worldwide incidence of new TB cases has been progressively falling for the last decade but the recent years has markedly changed this trend due to the advent of AIDS and the appearance of multidrug resistant strains of
M. tuberculosis.
The only vaccine presently available for clinical use is BCG, a vaccine which efficacy remains a matter of controversy. BCG generally induces a high level of acquired resistance in animal models of TB, but several human trials in developing countries have failed to demonstrate significant protection. Notably, BCG is not approved by the FDA for use in the United States.
This makes the development of a new and improved vaccine against TB an urgent matter which has been given a very high priority by the WHO. Many attempts to define protective mycobacterial substances have been made, and from 1950 to several investigators reported an increased resistance after experimental vaccination. However, the demonstration of a specific long-term protective immune response with the potency of BCG has not yet been achieved by administration of soluble proteins or cell wall fragments, although progress is currently being made by relying on polypeptides derived from short term-culture filtrate, cf. the discussion below.
Immunity to
M. tuberculosis
is characterized by three basic features; i) Living bacilli efficiently induces a protective immune response in contrast to killed preparations; ii) Specifically sensitized T lymphocytes mediate this protection; iii) The most important mediator molecule seems to be interferon gamma (INF-&ggr;). Short term-culture filtrate (ST-CF) is a complex mixture of proteins released from
M. tuberculosis
during the first few days of growth in a liquid medium (Andersen et al., 1991). Culture filtrates has been suggested to hold protective antigens recognized by the host in the first phase of TB infection (Andersen et al. 1991, Orme et al. 1993). Recent data from several laboratories have demonstrated that experimental subunit vaccines based on culture filtrate antigens can provide high levels of acquired resistance to TB (Pal and Horwitz, 1992; Roberts et al., 1995; Andersen, 1994; Lindblad et al., 1997). Culture filtrates are, however, complex protein mixtures and until now very limited information has been available on the molecules responsible for this protective immune response. In this regard, only two culture filtrate antigens have been described as involved in protective immunity, the low mass antigen ESAT-6 (Andersen et al., 1995 and EP-A-0 706 571) and the 31 kDa molecule Ag85B (EP-0 432 203).
There is therefore a need for the identification of further antigens involved in the induction of protective immunity against TB in order to eventually produce an effective sub-unit vaccine.
OBJECT OF THE INVENTION
It is an object of the invention to provide novel antigens which are effective as components in a subunit vaccine against TB or which are useful as components in diagnostic compositions for the detection of infection with mycobacteria, especially virulence-associated mycobacteria. The novel antigens may also be important drug targets.
SUMMARY OF THE INVENTION
The present invention is i.a. based on the identification and characterization of a number of previously uncharacterized culture filtrate antigens from
M. tuberculosis.
In animal models of TB, T cells mediating immunity are focused predominantly to antigens in the regions 6-12 and 17-30 kDa of STCF. In the present invention 8 antigens in the low molecular weight region (CFP7, CFP7A, CFP7B, CFP8A, CFP8B, CFP9, CFP10A, and CFP11) and 18 antigens (CFP16, CFP17, CFP19, CFP19B, CFP20, CFP21, CFP22, CFP22A, CFP23, CFP23A, CFP23B, CFP25, CFP26, CFP27, CFP28, CFP29, CFP30A, and CFP30B) in the 17-30 kDa region have been identified. Of these, CFP19A and CFP23 have been selected because they exhibit relatively high homologies with CFP21 and CFP25, respectively, in so far that a nucleotide homology sequence search in the Sanger Database (cf. below) with the genes encoding CFP21 and CFP25, (cfp25 and cfp21 respectively), shows homology to two
M. tuberculosis
DNA sequences, orf19A and orf23. The two sequences, orf19a and orf23, encode to putative proteins CFP19A and CFP23 with the molecular weights of approx. 19 and 23 kDa respectively. The identity, at amino acid level, to CFP21 and CFP25 is 46% and 50%, respectively, for both proteins. CFP21 and CFP25 have been shown to be dominant T-cell antigens, and it is therefore believed that CFP19A and CFP23 are possible new T-cell antigens.
Furthermore, a 50 kDa antigen (CFP50) has been isolated from culture filtrate and so has also an antigen (CWP32) isolated from the cell wall in the 30 kDa region.
The present invention is also based on the identification of a number of putative antigens from
M. tuberculosis
which are not present in
Mycobacterium bovis
BCG strains. The nucleotide sequences encoding these putative antigens are:
rd1-orf2, rd1-orf3, rd1-orf4, rd1-orf5, rd1-orf8, rd1-orf9a, and rd1-orf9b.
Finally, the invention is based on the surprising discovery that fusions between ESAT-6 and MPT59 are superior immunogens compared to the unfused proteins, respectively.
The encoding genes for 33 of the antigens have been determined, the distribution of a number of the antigens in various mycobacterial strains investigated and the biological activity of the products characterized. The panel hold antigens with potential for vaccine purposes as well as for diagnostic purposes, since the antigens are all secreted by metabolizing mycobacteria.
The following table lists the antigens of the invention by the names used herein as well as by reference to relevant SEQ ID NOs of N-terminal sequences, full amino acid sequences and sequences of DNA encoding the antigens:
N-terminal
Nucleotide
Amino acid
sequence
sequence
sequence
Antigen
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
CFP7
1
2
CFP7A
81
47
48
CFP7B
168
146
147
CFP8A
73
148
149
CFP8B
74
150
151
CFP9
3
4
CFP10A
169
140
141
CFP11
170
142
143
CFP16
79
63
64
CFP17
17
5
6
CFP19
82
49
50
CFP19A
51
52
CFP19B
80
CFP20
18
7
8
CFP21
19
9
10
CFP22
20
11
12
CFP22A
83
53
54
CFP23
55
56
CFP23A
76
CFP23B
75
CFP25
21
13
14
CFP25A
78
65
66
CFP27
84
57
58
CFP28
22
CFP29
23
15
16
CFP30A
85
59
60
CFP30B
171
144
145
CFP50
86
61
62
MPT51
41
42
CWP32
77
152
153
RD1-ORF8
67
68
RD1-ORF2
71
72
RD1-ORF9B
69
70
RD1-ORF3
87
88
RD1-ORF9A
93
94
RD1-ORF4
89
90
RD1-ORF5
91
92
MPT59-
172
ESAT6
ESAT6-
173
MPT59
It is well-known in the art that T-cell epitopes are responsible for the elicitation of the acquired immunity against TB, whereas B-cell epitopes are without any significant influence on acquired immunity and recognition of mycobacteria in vivo. Since such T-cell epitopes are linear and are known to have a minimum length of 6 amino acid residues, the present invention is especially concerned with the identification and utilisation of such T-cell epitopes.
Hence, in its broadest aspect the invention relates to a substantially pure polypeptide fragment which
a) comprises an amino acid sequence selected from the sequences shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, any one of 17-23, 42, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, any one of 72
Andersen Peter
Florio Walter
Nielsen Rikke
Oettinger Thomas
Rasmussen Peter Birk
Frommer Lawrence & Haug
Kowalski Thomas J.
Statens Serum Institut
Swartz Rodney P
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