Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
1999-12-21
2002-10-29
McGarry, Sean (Department: 1635)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S006120, C435S091100, C435S320100, C536S023100, C536S023700
Reexamination Certificate
active
06472213
ABSTRACT:
This invention relates to shuttle vectors for inserting DNA in mycobacteria.
It also relates to the use as vaccines of these mycobacteria in which one of these vectors has been inserted.
The Mycobacterium genus comprises over 50 species, including major pathogens for the human such as
Mycobacterium tuberculosis
, agent of tuberculosis,
Mycobacterium leprae
, agent of leprosy, and
Mycobacterium avium
, a major opportunist in the course of AIDS. Little is known of the genetic mechanisms of the virulence of
M. tuberculosis
and the other pathogenic species for the human, due to the low growth rate of these bacteria and the lack of adequate cloning vectors; and as a result it is difficult to insert foreign DNA in these microorganisms. Moreover, the mycobacteria show a particular resistance to many antibiotics due to the fact that the constitution of their wall is poorly permeable to these molecules. However, recent studies have shown that it is possible to introduce foreign DNA in BCG with a view to expressing antigens heterologous to this bacterium.
In the case of BCG manipulation, the use of markers of resistance to antibiotics hitherto available in mycobacteria such as the aph3 gene providing a high degree of resistance to kanamycin or genes providing resistance to streptomycin or spectinomycin, cannot be envisaged because it could cause a dissemination of resistance to these antibiotics in the environment in the event that recombinant BCG is used for therapeutic or prophylactic purposes in the human or the animal. It is thus of the utmost importance to be able to develop resistance markers which are harmless for the environment in order to select recombinant mycobacteria which can be widely used.
At the present time, only a very small number of markers usable in mycobacteria is available.
Thus, Aldovini et al. (J. Bacteriol, 1993, 175, 7282-7289) have isolated the gene coding for orotidine-5′-monophosphate decarboxylase (OMP-DCase) of
Mycobacterium bovis
BCG.
Another marker, the cI gene, coding for the L1 phage repressor, has also been used (patent application WO-90/00594).
Shuttle vectors based on these markers and allowing the transfer of DNA between
Escherichia coli
and Mycobacterium have already been described. Thus, patent application WO-91/13 157 describes two shuttle vectors, pEP2 and pEP3, respectively carriers of a gene of resistance to kanamycin and hygromycin.
One of these vectors, pEP2, is modified by the introduction of the protein promoter MBP70 of Mycobacterium, as described in patent application WO-90/10 701.
Other shuttle vectors usable in mycobacteria and carrying markers of resistance to antibiotics, auxotrophic markers or the cI gene have been described in patent application WO 90/00594.
Finally, Ranes et al. (J. Bacteriol. 1990, 172, 2793-2797) have described the construction of a shuttle vector called pRR3 carrying a gene of resistance to kanamycin expressing itself in
Mycobacterium bovis
BCG and
Mycobacterium smegmatis.
A strain of non-pathogenic saprophytic mycobacterium resistant to mercury carrying a plasmid of 115 MD was also known (Meissner and Falkinham, J. Bacteriol, 157, 669-672, 1984). This very large plasmid was not, however, characterized. In particular, the genes involved in the resistance to mercury were neither localized nor identified, and consequently this plasmid could not be used as a shuttle plasmid. Moreover, saprophytic mycobacteria do not belong to pathogenic mycobacteria, the latter being principally included in the
M. tuberculosis, M. bovis
and
M. leprae species.
The result was that those skilled in the art wishing to transfer DNA coding for antigens, obtained by cloning in
Escherichia coli
, to mycobacteria with a view to fabricating vaccines, was confronted with a lack of easy-to-manipulate vectors carrying markers expressing themselves in
E. coli
and mycobacteria, and without danger to human or animal health. The use of genes of resistance to antibiotics of therapeutic aim must, in fact, be ruled out for the reasons given above. The use of auxotrophic mutants gives rise to practical difficulties of selection and moreover involves the need to obtain mutants for each mycobacterium strain.
The applicants therefore set out to find other markers which could be included in shuttle vectors without excessively reducing the ability of said vectors to include other DNA fragments, and which would provide on the mycobacteria in which they are expressed characters making it possible to distinguish them, in a way which is both unambiguous and easy to implement, from mycobacteria not carrying these markers.
The applicants have thus surprisingly revealed that shuttle vectors originating from Gram-negative bacteria, carrying genes of resistance to a compound containing a heavy metal such as inorganic mercury or. mercurial compounds, were expressed in Gram-positive bacteria and could be used for the transfer of DNA between
Escherichia coli
and Mycobacterium, and that bacteria carrying these shuttle vectors could be selected easily.
This invention therefore relates to a shuttle vector for inserting DNA in mycobacteria comprising at least one origin of functional replication in said mycobacteria, another origin of functional replication in other bacteria, such as
E. coli
, an enzyme cutting site allowing the insertion of DNA coding for a protein capable of being expressed in the mycobacteria, characterized in that it also carries a gene providing on said mycobacteria resistance to a compound containing a heavy metal such as inorganic mercury, a mercurial compound or arsenic.
It should be considered that those skilled in the art have for many years been confronted with the problem of seeking markers for the transfer of genes in mycobacteria, but that up to now no satisfactory solution has been found to this problem.
The solution found by the applicants, namely the choice of genes originating from Gram-negative bacteria and coding for a resistance to a heavy metal, is particularly surprising in view of the fact that those skilled in the art would not in theory have expected to find Gram-negative bacteria expressed in Gram-positive bacteria. Indeed, many studies have demonstrated the inability of genes from Gram-negative bacteria to express themselves in Gram-positive bacteria (Misra, Plasmid, 27, 4-16, 1992; Robinson and Tuovinen, Microbiological Reviews, Vol. 48 No. 2, 95-124, 1984).
The applicants have thus found a solution to a long-standing problem and one which, moreover, is of great importance to public health: mycobacteria and in particular
M. bovis
BCG, are considered as good candidates for vaccination against various diseases, provided that it is possible for these bacteria to express foreign antigens. This invention provides for an introduction of genes coding for these antigens which is both efficient and without danger to human health.
This invention therefore involves a major technical advance in the field of public health.
It should also be noticed that although markers of resistance to mercury were already known to exist, particularly in mycobacteria, those skilled in the art were in no way incited to integrate genes of resistance to mercury or mercurial compounds in shuttle vectors usable in mycobacteria. On the contrary, it is widely known that operons expressed for example in
E. coli
are not expressed in mycobacteria. Those skilled in the art were thus not incited to use in mycobacteria genes usually expressed in
E. coli.
In other words, nothing suggested using genes of resistance, in particular to mercury or mercurial compounds, as genes of selection of mycobacteria having received the shuttle vectors.
While one of the aims of this invention relates to the transfer of heterologous DNA to Mycobacterium, it should be noted that the shuttle vectors may also be used for transferring DNA fragments of mycobacteria to other bacteria such as
Escherichia coli
in order to facilitate the genetic study of the mycobacteria. It is a fact that mycobacteria, largely due to their slow growth rate, are difficult
Baulard Alain
Berche Patrick
Escuyer Vincent
Haddad Nadia
Locht Camille
Institut National de la Sante et de la Recherche Medicale (Inser
McGarry Sean
Zara J.
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