Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Recombinant or stably-transformed bacterium encoding one or...
Reexamination Certificate
2000-02-22
2004-01-06
Swartz, Rodney P (Department: 1645)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Recombinant or stably-transformed bacterium encoding one or...
C424S009100, C424S009200, C424S184100, C424S185100, C424S190100, C424S192100, C424S203100, C424S234100, C424S248100, C424S278100, C424S093100, C424S093200, C435S320100, C530S300000, C530S350000, C536S023100, C536S023700
Reexamination Certificate
active
06673353
ABSTRACT:
The present invention relates to novel recombinant vaccines providing protective immunity especially against tuberculosis. Further, the present invention refers to novel recombinant nucleic acid molecules, vectors containing said nucleic acid molecules, cells transformed with said nucleic acid molecules and polypeptides encoded by said nucleic acid molecules.
Tuberculosis (TB) caused by
Mycobacterium tuberculosis
remains a significant global problem. It is estimated that one third of the world's population is infected with
M.tuberculosis
(Kochi, 1991). In many countries the only measure for TB control has been vaccination with
M.bovis
bacille Calmette-Guérin (BCG). The overall vaccine efficacy of BCG against TB, however, is about 50% with extreme variations ranging from 0% to 80% between different field trials (Roche et al., 1995). Thus, BCG should be improved, e.g. by genetic engineering, to provide a vaccine for better TB control (Murray et al., 1996; Hess and Kaufmann, 1993). The widespread emergence of multiple drug-resistant
M.tuberculosis
strains additionally underlines the urgent requirement for novel TB vaccines (Grange, 1996).
M.tuberculosis
belongs to the group of intracellular bacteria that replicate within the phagosomal vacuoles of resting macrophages, thus protection against TB depends on T cell-mediated immunity (Kaufmann, 1993). Several studies in mice and humans, however, have shown that mycobacteria stimulate antigen-specific, major histocompatibility complex (MHC) class II- or class I-restricted CD4 and CD8 T cells, respectively (Kaufmann, 1993).
The important role of MHC class I-restricted CD8 T cells was convincingly demonstrated by the failure of &bgr;2-microglobulin (&bgr;2m) deficient mice to control experimental
M.tuberculosis
infection (Flynn et al., 1993). Because these mutant mice lack MHC class I, functional CD8 T cells cannot develop. In contrast to
M.tuberculosis
infection, &bgr;2m-deficient mice are capable of controlling certain infectious doses of the BCG vaccine strain (Flynn et al., 1993; Ladel et al., 1995). Furthermore, BCG vaccination of &bgr;2m-deficient mice prolonged survival after subsequent
M.tuberculosis
infection whereas BCG-immunized C57BL/6 resisted TB (Flynn et al., 1993). This differential CD8 T cell dependency between
M.tuberculosis
and BCG may be explained as follows:
M.tuberculosis
antigens gain better access to the cytoplasm than antigens from BCG leading to more pronounced MHC class I presentation (Hess and Kaufmann, 1993). Consequently, a more effective CD8 T cell response is generated by
M.tuberculosis.
This notion was recently supported by increased MHC class I presentation of an irrelevant antigen, ovalbumin, by simultaneous
M.tuberculosis,
rather than BCG, infection of antigen presenting cells (APC) (Mazzaccaro et al., 1996).
Secreted proteins of
M.tuberculosis
comprise a valuable source of antigens for MHC class I presentation. Recently, a DNA vaccine encoding the secreted antigen Ag85A elicited MHC class I-restricted CD8 T cell responses in mice which may contribute to defence against TB (Huygen et al., 1996). In general, evidence is accumulating that immunization with secreted protein antigens of
M.tuberculosis
induce some protection against TB in guinea pigs and mice (Horwitz et al., 1995; Andersen, 1994). An important goal towards the development of improved TB vaccines based on BCG, therefore, is to augment the accessibility of secreted BCG-specific antigens to the cytoplasm of infected APC. Subsequent delivery of peptides derived from these secreted proteins into the MHC class I presentation pathway may potentiate the already existing BCG-specific immune response for preventing TB.
The phagolysosomal escape of
L.monocytogenes
represents a unique mechanism to facilitate MHC class I antigen presentation of listerial antigens (Berche et al., 1987; Portnoy et al., 1988). Listeriolysin (Hly), a pore-forming sulfhydryl-activated cytolysin, is essential for the release of
L.monocytogenes
microorganisms from phagolysosomal vacuoles into the cytosol of host cells (Gaillard et al., 1987; Portnoy et al., 1988). This escape function was recently transferred to
Bacillus subtilis
and to attenuated Salmonella ssp. strains (Bielecki et al., 1991; Gentschev et al., 1995; Hess and Kaufmann, 1997). Hly expression by an asporogenic
B.subtilis
mutant strain or in Salmonella ssp. results in bacterial escape from the phagolysosome into the cytosol of J774 macrophage-like cells (Bielecki et al., 1991; Gentschev et al., 1995; Hess and Kaufmann, 1997).
Thus, the transfer of lysosomal escape functions to heterologous microorganisms may cause an elevated toxicity of the resulting recombinant microorganisms. For this reason, the use of these lysosomal escape functions for the preparation of recombinant living vaccines has not been readily taken into consideration.
According to the present invention recombinant BCG strains secreting hemolytically active Hly were constructed which show an improved efficacy MHC class I-restricted immune response and, surprisingly, an equal or even lower cytotoxicity in comparison with the unmodified native BCG strains. Thus, these recombinant organisms are promising candidate vaccines against TB.
A first aspect of the present invention is a recombinant nucleic acid molecule encoding a fusion polypeptide comprising (a) at least one domain from a Mycobacterium polypeptide, wherein said domain is capable of eliciting an immune response in a mammal, and (b) a phagolysosomal escape domain.
A specific embodiment of this first aspect is the nucleic acid molecule in SEQ ID No.1. This nucleic acid molecule comprises a signal peptide coding sequence (nucleotide 1-120), a sequence coding for an immunogenic domain (nucleotide 121-153), a peptide linker coding sequence (nucleotide 154-210), a sequence coding for a phagolysosomal domain (nucleotide 211-1722), a further peptide linker coding sequence (nucleotide 1723-1800) and a sequence coding for a random peptide (nucleotide 1801-1870). The corresponding amino acid sequence is shown in SEQ ID No.2.
The nucleic acid of the present invention contains at least one immunogenic domain from a polypeptide derived from an organism of the genus Mycobacterium, preferably from
Mycobacterium tuberculosis
or from
Mycobacterium bovis.
This domain has a length of at least 6, preferably of at least 8 amino acids. The immunogenic domain is preferably a portion of a native Mycobacterium polypeptide. However, within the scope of the present invention is also a modified immunogenic domain, which is derived from a native immunogenic domain by substituting, deleting and/or adding one or several amino acids.
The immunogenic domain is capable of eliciting an immune response in a mammal. This immune response can be a B cell-mediated immune response. Preferably, however, the immunogenic domain is capable of eliciting a T cell-mediated immune response, more preferably a MHC class I-restricted CD8 T cell response.
The domain capable of eliciting an immune response is peferably selected from immunogenic peptides or polypeptides from
M.bovis
or
M.tuberculosis
or from immunogenic fragments thereof. Specific examples for suitable antigens are Ag85B (p30) from
M.tuberculosis
(Harth et al., 1996), Ag85B (&agr;-antigen) from
M.bovis
BCG (Matsuo et al., 1988), Ag85A from
M.tuberculosis
(Huygen et al., 1996) and ESAT-6 from
M.tuberculosis
(Sorensen et al., 1996, Harboe et al., 1996 and Andersen et al., 1995). More preferably, the immunogenic domain is derived from the antigen Ag85B. Most preferably, the immunogenic domain comprises the sequence from aa.41 to aa.51 in SEQ ID No.2.
The recombinant nucleic acid molecule according to the present invention further comprises a phagolysosomal escape domain, i.e. a polypeptide domain which provides for an escape of the fusion polypeptide from the phagolysosome into the cytosol of mammalian cells. Preferably, the phagolysosomal escape domain is derived from an organism of the genus Listeria. More preferably, th
Hess Jürgen
Kaufmann Stefan H. E.
Max-Planck-Gesellschaft zur Forderung der Wissenschaften e.V.
Rothwell Figg Ernst & Manbeck
Swartz Rodney P
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