Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Bacterium or component thereof or substance produced by said...
Reexamination Certificate
1997-12-01
2001-10-02
Minnifield, Nita (Department: 1645)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Bacterium or component thereof or substance produced by said...
C424S234100, C424S235100, C424S190100, C424S184100, C424S197110, C424S825000, C530S350000, C530S300000, C530S324000
Reexamination Certificate
active
06296855
ABSTRACT:
The present invention relates to an isolated and pure 17-kDa
Brucella abortus
antigen, which can be used for the diagnosis of Brucella infection in human and cattle. The invention also relates to nucleic acids coding for said antigen, as well as to diagnostic methods and kits using such nucleic acids for detecting Brucella infection. The invention also relates to recombinant polypeptides, a process for preparing the same and their use in methods and kits for the diagnosis of Brucella infection. The invention also relates to the possible use of said isolated antigen or said recombinant polypeptides as an active principle of a vaccine composition against Brucella strains. The invention relates also to a vaccine composition comprising a recombinant Brucella strain, specifically deleted for the gene encoding said antigen.
Brucellosis is an infection due to a small intracellular gram-negative bacterium which is pathogenic for humans as well as for domestic animals. This infection induces abortions in livestock animals leading to severe economic losses. Within the genus Brucelia, six closely related species have been described (Fekete et al., 1992; Verger et al., 1985; Verstraete & Winter, 1984), the most important of which are
B. abortus
and
B. melitensis
. Humans and ruminants (sheep, goats and cows) are predominantly infected by these two Brucelia strains. Serological tests currently used for diagnosis of brucellosis infection are based on the detection of anti-lipopolysaccharide (LPS) antibodies (Alton et al., 1988). These tests do not permit the differentiation between vaccinated and infected animals, and fail to reveal some infected animals which are positive in an intradermic test. Moreover important cross-reactions with other gram-negative bacteria have been reported (Corbel et al., 1983; Perry & Bundle, 1990; Schoerner et al., 1990). Diagnostic tests with higher specificity are based on the isolation of Brucella bacteria or on the intradermic injection of a protein preparation from Brucella bacteria “Brucellergen” (Fensterbank, 1984), leading to a delayed type hypersensitivity reaction (DTH). However, classical bacteriology is time-consuming and “Brucellergen” preparations are not always easy to produce free of LPS. The latter can cause seroconversion of animals upon DTH testing, precluding its serological distinction from infected animals. The identification of specific antigens for brucellosis diagnosis is therefore a matter of great interest for the development of a specific serological test.
For prophylactic vaccination against brucellosis, today two live vaccine strains are being used succesfully. The B19 strain is mostly used in cattle and the Rev. 1 strain in small ruminants. The H38 killed vaccine has also been used. Although good protection is generally obtained with these vaccines, the general drawback is the induction of an immune response in the vaccinated animals, which precludes the distinction between infected and vaccinated animals.
A
Brucella abortus
cytoplasmic preparation was described by Goldbaum et al. (1993) which showed several bands of different molecular weights. The two major components are those of 18- and 36-kDa. This cytoplasmic preparation was obtained by immunoadsorption of a
B. abortus
cytoplasmic fraction with an IgG2b monoclonal antibody (BI24) produced by immunizing with a LPS-free cytoplasmic antigenic fraction preparation (LPS-free CYT) as antigen. Microsequencing of the identified cytoplasmic preparation (including the 18- and 36-kDa bands) resulted in the elucidation of the sequence of only the following three tryptic peptides:
(i) SEQ ID NO: 6
(ii) SEQ ID NO: 7
(iii) SEQ ID NO: 8
Another cytoplasmic Brucella protein preparation with an apparent molecular mass of 20 kDa has been described by Zygmunt et al. (1992).
Cloeckaert et al. (1990, 1991) describe the production of 26 anti-Brucella OMP monoclonal antibodies to R and S Brucella cells directed against seven outer membrane protein components.
The aim of the present invention is to provide a Brucella antigen or a polynucleic acid encoding the same which is useful for diagnosing in vitro Brucella infection (=brucellosis) in mammals (humans, ruminants).
A special aim of the invention is to provide a Brucella antigen or a polynucleic acid encoding the same which is useful for differentiating between field infected and vaccinated individuals.
Another aim of the present invention is to provide purified and isolated 17 kDa antigen of Brucelia, and more particularly purified and isolated
B. abortus
17 kDa antigen.
Another aim of the present invention is to provide amino acid and corresponding nucleotide sequences of said Brucella 17 kDa antigen.
Another aim of the present invention is to provide antibodies specifically directed against said Brucella 17 kDa antigen.
Another aim of the present invention is to provide primers and probes derived from the nucleotide sequences encoding said Brucella 17 kDa antigen.
Another aim of the present invention is to provide diagnostic methods or kits for diagnosing Brucella infection, more particularly for differentiating between field infected and vaccinated individuals, based on any of the above-mentioned polypeptides or polynucleic acids as active compounds.
Another aim of the present invention is to provide vaccine compositions providing protective immunity towards brucellosis in mammals (humans, ruminants).
Another aim of the present invention is to provide a recombinant Brucella strain in which the gene encoding the Brucella 17 kDa antigen has been deleted or inactivated.
The present invention relates more particularly to an isolated 17-kDa Brucella antigen characterized by an amino acid sequence having at least 60% homology, preferably at least 70% homology, more preferably at least 80% homology to the 158 residue amino acid sequence as shown in SEQ ID NO 2, or fragments of said antigen, consisting of at least 9 contiguous amino acids from said amino acid sequence.
The term “isolated” refers to a purity grade of at least 90%, preferably 95% and more preferably of 98% of the antigen expressed in weight versus contaminants, as determined by one or two dimensional SDS-PAGE. Said purity may be obtained by purification of the naturally occurring polypeptide, or by de novo synthesis of the polypeptide, by chemical methods or by recombinant DNA technology, and subsequent purification. The term “isolated” thus implies that the antigen is in a different state and environment than the naturally occurring antigen.
The word “antigen” refers to a molecule which provokes an immune response (also called “immunogen”), or which can be recognized by the immune system (also called “antigen sensu strictu”). The immune response or the immune recognition reaction can be of the cellular or humoral type.
The term “17 kDa antigen” refers to an antigen having a molecular weight of approximately 17 kDa as determined by SDS-PAGE. Said determined molecular weight may vary according to strain to strain variations, or according to methodology variations. Preferably, the molecular weight of the antigen of the invention as determined by SDS-PAGE is between 14 and 20 kDa, more preferably between 15 and 19 kDa, most preferably between 16 and 18 kDa.
The terms “homologous” and “homology” are used in the current invention as synonyms for “identical” and “identity”; this means that amino acid sequences which are e.g. said to be 55% homologous, show 55% identical amino acids in the same position upon alignment of the sequences.
In an effort to identify a purified and isolated Brucella 17-kDa antigen, an expression library containing
B. abortus
chromosomal DNA inserts was screened with sera from field infected sheep (see Examples section). The DNA insert from a positive recombinant phage was analyzed in detail. Sequence analysis revealed a new gene encoding a 17-kDa
Brucella abortus
protein (SEQ ID NO 1). The gene was recombinantly expressed, and the antigenicity of the recombinantly produced new protein was analyzed in Western blotting, in ELISA using sera from in
Hemmen Fabienne
Saman Eric
Howrey Simon Arnold & White , LLP
Innogenetics N.V.
Minnifield Nita
LandOfFree
17-KDA Brucella abortus antigen, recombinant polypeptides,... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with 17-KDA Brucella abortus antigen, recombinant polypeptides,..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and 17-KDA Brucella abortus antigen, recombinant polypeptides,... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2582874