Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
1997-06-09
2001-06-19
Swartz, Rodney P. (Department: 1641)
Chemistry: molecular biology and microbiology
Vector, per se
C435S183000, C530S387100, C536S023700, C536S024100, C536S024320
Reexamination Certificate
active
06248581
ABSTRACT:
The Mycobacterium genus includes major human pathogens such as
M. leprae
and
M. tuberculosis,
the agents responsible for leprosy and tuberculosis, which remain serious public health problems world-wide.
M. bovis
and
M. tuberculosis,
the causative agents of tuberculosis, are intracellular facultative bacteria. Despite the major health problems linked to these pathogenic organisms, little is known about their exported and/or secreted proteins. In SDS-PAGE analyses of
M. tuberculosis
culture filtrate show at least 30 secreted proteins (1,19,38). Some of them have been characterized, their genes cloned and sequenced (7, 35, 37). Others, although they are immunodominant antigens of major importance for inducing protective immunity (2, 21), have not been completely identified. In addition, it is probable that a great number of exported proteins remain attached to the cell membrane and, consequently, are not present in culture supernatants. It has been shown that proteins located at the outer surface of various pathogenic bacteria, such as the 103 kDa
Yersina pseudotuberculosis
invasin (14) or the 80 kDa
Listeria monocytogenes
internalin (10) play an important role in interactions with the host cells and, consequently, in pathogenicity as in the induction of protective responses. Thus, a membrane-bound protein could be important for
M. tuberculosis
infection as well as for the induction of a protective response against this infection. These proteins could certainly be of interest for the preparation of vaccines.
The BCG (Bacille CalmetteGuérin), an avirulent strain derived from
M. bovis,
has been widely used as vaccine against tuberculosis. It is also a very important vector for the construction of live recombinant vaccines, particularly because of its high immunogenicity. Consequently, the study of the molecular biology of mycobacteria is currently of great interest.
The development of new vaccines against pathogenic mycobacteria, or the improvement of available vaccines required the development of specific tools which make it possible to isolate or obtain immunogenic polypeptide sequences.
The inventors have defined and produced, for this purpose, new vectors allowing the screening of mycobacteria DNA sequences in order to identify, among these sequences, nucleic acids encoding proteins of interest.
Vectors have been defined for evaluating the efficacy of sequences for regulation of expression in mycobacteria.
The invention also relates to new mycobacteria polypeptides which may have been isolated by means of the preceding vectors and capable of entering into the production of compositions for the detection of a mycobacteria infection, or for protection against an infection due to mycobacteria.
The subject of the invention is therefore a recombinant screening and/or cloning and/or expression vector, characterized in that it replicates in mycobacteria, in that it contains
1) a replicon which is functional in mycobacteria;
2) a selectable marker;
3) a reporter cassette comprising
a) a multiple cloning site (polylinker),
b) a transcription terminator which is active in mycobacteria, upstream of the polylinker, and
c) a coding nucleotide sequence derived from a gene encoding a marker for expression and/or export and/or secretion of protein, said nucleotide sequence lacking its initiation codon and its regulatory sequences.
The marker for export and/or secretion is a nucleotide sequence whose expression followed by export and/or secretion depends on regulatory elements which control its expression.
“Sequences or elements for regulation of expression” is understood to mean a promoter sequence for transcription, a sequence comprising the ribosome-binding site (RBS), the sequences responsible for export and/or secretion such as the sequence termed signal sequence.
A first advantageous marker for export and/or expression is a coding sequence derived from the PhoA gene. Where appropriate, it is truncated such that the alkaline phosphatase activity is, nevertheless, capable of being restored when the truncated coding sequence is placed under the control of a promoter and of appropriate regulatory elements.
Other markers for exposure and/or export and/or secretion may be used. There may be mentioned by way of examples a sequence of the gene for &bgr;-agarase or for nuclease of a staphylococcus or for &bgr;-lactamase of a mycobacterium.
The transcription terminator should be functional in mycobacteria. An advantageous terminator is, in this regard, the T4 coliphage terminator (tT4). Other terminators appropriate for carrying out the invention may be isolated using the technique presented in the examples, for example by means of the vector pJN3.
A vector which is particularly preferred for carrying out the invention is the plasmid pJEM11 deposited at CNCM (Collection Nationale de Cultures de Microorganismes in Paris—France) under the No. I-1375, on Nov. 3, 1993.
For the selection or the identification of mycobacteria nucleic acid sequences encoding products capable of being incorporated into immunogenic or antigenic compositions for the detection of a mycobacteria infection, the vector of the invention will comprise, in one of the polylinker sites, a nucleotide sequence from a mycobacterium in which the presence of regulatory sequences is being sought which are associated with all or part of a gene of interest making it possible, when the vector carrying these sequences (recombinant vector), is intergrated or replicates in a mycobacterium-type cellular host, to obtain the exposure at the level of the cell wall or membrane of the host, and/or export and/or secretion of the product of expression of the abovementioned nucleotide sequence.
The mycobacteria sequence in question may be any sequence for which attempts are made to detect if it contains elements for regulation of expression associated with all or part of a gene of interest and capable of allowing or promoting exposure at the level of the cell membrane of a host in which it might be expressed, and/or export and/or secretion of a product of expression of a given coding sequence and, by way of test, of the marker for export and/or secretion.
Preferably, this sequence is obtained by enzymatic digestion of the genomic DNA or of the DNA complementary to an RNA of a mycobacterium and preferably of a pathogenic mycobacterium.
According to a first embodiment of the invention, the enzymatic digestion of the genomic DNA or of the complementary DNA is carried out using
M. tuberculosis.
Preferably, this DNA is digested with an enzyme such as sau3A.
Other digestive enzymes such as ScaI, ApaI, ScaII, KpnI or alternatively exonucleases or polymerases, may naturally be used, as long as they allow fragments to be obtained whose ends may be inserted into one of the cloning sites of the polylinker of the vector according to the invention.
Where appropriate, digestions with different enzymes will be carried out simultaneously.
Preferred recombinant vectors for carrying out the invention are chosen among the following recombinant vectors deposited at CNCM on Aug. 8, 1994:
pExp53 deposited at CNCM under the No. I-1464
pExp59 deposited at CNCM under the No. I-1465
pExp410 deposited at CNCM under the No. I-1466
pExp421 deposited at CNCM under the No. I-1467.
The vectors of the invention may also be used to determine the presence of sequences of interest, according to what was stated above, in mycobacteria such as
M. africanum, M. bovis, M. avium
or
M. leprae
whose DNA or cDNA will have been treated with determined enzymes.
The subject of the invention is also a process for screening nucleotide sequences derived from mycobacteria, to determine the presence, in these sequences, of regulatory elements controlling the expression, in a cellular host, of nucleic acid sequences containing them, and/or exposure at the surface of the cellular host and/or export and/or secretion of the polypeptide sequences resulting from the expression of the abovementioned nucleotide sequences, characterized in that it comprises the following steps:
a) digestion
Berthet Francois-Xavier
Gicquel Brigitte
Lim Eng Mong
Portnoi Denis
Timm Juliano
Finnegan Henderson Farabow Garrett & Dunner L.L.P.
Institut Pasteur
Swartz Rodney P.
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