Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Recombinant or stably-transformed bacterium encoding one or...
Reexamination Certificate
2000-05-10
2003-07-01
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, C424S130100, C424S150100, C424S164100, C424S168100, C424S184100, C424S185100, C424S248100, C530S300000, C530S350000, C536S023100, C536S023700
Reexamination Certificate
active
06585976
ABSTRACT:
The invention relates to a Mycobacterium strain with modified erp gene and the vaccine composition containing same.
Tuberculosis is an infectious disease caused in most cases by inhalation of bacteria belonging to the complex of
Mycobacterium tuberculosis
species (
M. africanum, M. bovis, M. tuberculosis
). With eight million new human cases annually causing three million deaths worldwide, tuberculosis remains a major public health problem (Sudre et al., 1992). The discovery of effective antibiotics (streptomycin, isoniazide, rifampicin and the like) appeared to allow the eradication of this disease. However, it is estimated that currently only 50% of patients are diagnosed and receive treatment. This treatment is often inappropriate or poorly monitored and leads to the appearance of an increasing number of antibiotic-resistant and even polychemoresistant strains (Dooley et al., 1992). In this context, the development of a vaccinal prophylaxis appears as a preferred solution for the control and eradication of tuberculosis.
The fact that an attenuated pathogenic bacterium is used as a component of a vaccine has been widely described and implemented in the prior art. The methods for obtaining such attenuated bacteria comprise the random selection of mutants induced chemically or by irradiation, or the production of recombinant bacteria of pathogenic origin in which a gene involved in a metabolic pathway has been inactivated by genetic engineering.
Straley et al. (1984) have studied the survival of avirulent mutants of
Yersinia pestis
which are deficient in one or more metabolic pathways.
Noriega et al. (1994) have manufactured, by genetic engineering, an oral Shigella strain intended to be used as a vaccine prototype by introducing deletions into a gene (aroA) encoding a protein involved in a metabolic pathway for an aromatic amino acid and they have demonstrated that the resulting defective recombinant Shigella strains were capable of inducing protective antibodies against the wild-type pathogen.
A major study has also been carried out using Salmonella as a model. See for example the reports by Hoiseth et al. (1981), Levine et al. (1987), Oyston et al. (1995) and Curtiss (1990).
However, similar studies have not yet been carried out for
Mycobacterium tuberculosis
, the etiological agent of tuberculosis (TB), which infects a third of the world population and kills three million people per year. Tuberculosis is the most important cause of mortality in the world caused by a group of infectious organisms (Bloom and Murray, 1992) grouped under the name “
M. tuberculosis
complex”. According to the WHO, more people died of tuberculosis in 1995 than during any previous year. It has been estimated that up to half a billion people will suffer from tuberculosis in the next 50 years. However, in spite of its importance, the genetic determinants of the virulence and persistance of
M. tuberculosis
remain scarcely characterized.
Indeed, the virulence of pathogenic mycobacteria is associated with their ability to grow and persist at the intracellular level. Bacteria of the
M. tuberculosis
complex parasitize the phagocytic cells in which they live and multiply in a specialized vacuolar compartment called the phagosome. The phagosomes containing live
M. tuberculosis
do not acidify and escape fusion with the lysosomes. The mechanisms by which
M. tuberculosis
make their phagosome more hospitable remain unknown and the mycobacterial genes affecting their intracellular growth and multiplication are being actively investigated.
The extreme difficulty of creating defined mutants of
M. tuberculosis
, either by allelic exchange or by transposon mutagenesis, has prevented the identification of these virulence factors according to the postulates of Koch (Falkow, 1988; Jacobs, 1992). Alternative genetic strategies have been used instead, including the complementation of a non-pathogenic bacterium (Arruda et al., 1993) and of spontaneous avirulent mutants with virulent
M. tuberculosis
(Pascopella et al., 1994) and virulent
M. bovis
(Collins et al., 1995) chromosomal DNA libraries. Although these studies have identified genes potentially involved in the entry into the epithelial cells and conferring a growth advantage in vivo, the great majority of the mycobacterial genes involved in the virulence and survival in the host organism remain unknown. The development of effective mutagene systems is therefore the priority for mycobacterial genetics.
One method for the creation of mutants is allelic exchange mutagenesis. Recently, allelic exchanges taking place with a low frequency have been demonstrated in bacteria of the
M. tuberculosis
complex using a suicide vector (Reyrat et al., 1995; Azad et al., 1996) and novel protocols allowing easier detection of the allelic exchange mutants have also been developed (Norman et al., 1995; Balasubramamian et al., 1996; Pelicic et al.,
FEMS Microbiol. Lett
. 1996). However, the detection of a very rare allelic exchange event is prevented by low transformation efficiencies and the high frequency of illegitimate recombinations. Thus, many Mycobacterium genes still remain refractory to allelic exchange by means of the available technologies.
More particularly, the allelic exchange mutagenesis systems require the use of more efficient methods. The problems encountered may be overcome by the use of a replicative vector which is effectively conditionally lost. The possibility of introducing such vectors makes it possible to avoid the problems resulting from the low transformation efficiencies. Thus, under counterselection conditions, the clones still containing the vector are eliminated, thus allowing the detection of very rare genetic events. Such a system has been recently developed. Using a replicative vector under certain conditions which is lost at 39° C. in
M. smegmatis
, the first library of mycobacterial insertion mutants was constructed in this rapidly growing model strain (Guilhot et al., 1994). However, the heat-sensitive vectors used are only slightly heat-sensitive in slow-growing mycobacteria of the
M. tuberculosis
complex and therefore cannot be used in these species for allelic exchange mutagenesis (unpublished data).
The inventors have succeeded in altering the virulence and the persistance of Mycobacterium strains in the host cells.
They have indeed produced a Mycobacterium strain one gene of which has been modified so as to attenuate its virulence.
Modified gene is understood to mean a gene which has undergone a modification abolishing the production of the corresponding protein or allowing the production of a protein which is at least 20% different in terms of activity compared with the natural protein.
BCG (Bacille Calmette-Guérin), an avirulent strain derived from
M. bovis
, is widely used worldwide as a vaccine against tuberculosis. However, while BCG can be administered without any problem to individuals with no immune deficiency, the same is not true for immunosuppressed individuals such as people infected with the AIDS virus, people who have had a marrow transplant, people suffering from a cancer, or people with altered functioning of one of the components of the immune system.
That is the reason why the present invention relates to a Mycobacterium strain with limited persistence.
The gene modified in the Mycobacterium strain in accordance with the invention is the erp gene. It may also be a gene having a complementation homology (of at least 80%) with the erp gene.
Analysis of the deduced protein sequence of the erp gene shows that the latter encodes a protein whose calculated molecular mass is 28 kDa. The presence of a signal sequence for export at an N-terminal position as well as the existence of a C-terminal hydrophobic region suggest that the molecule can be anchored in the plasma membrane or located at the surface of the bacilli. Furthermore, the central region of the protein comprises two repeat regions each composed of 6 copies of a P(G/A)LTS (SEQ ID NO: 1) motif positioned in tandem. This organization is similar to t
Berthet François-Xavier
Gicquel Brigitte
Finnegan Henderson Farabow Garrett & Dunner L.L.P.
Institut Pasteur
Swartz Rodney P
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