Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1996-06-28
2001-07-31
Longton, Enrique D. (Department: 1653)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S069700, C435S069800, C435S071100, C435S091200, C435S091400, C435S252300, C435S320100, C536S023100, C536S023700, C536S024330, C536S024320, C536S024100, C536S024300, C530S413000, C530S350000
Reexamination Certificate
active
06268171
ABSTRACT:
This application claims priority under 35 USC 119 to P 4,336,530.2 Fed. Rep. of Germany, filed Oct. 26, 1993, and PCT/EP94/03494, filed Oct. 25, 1994.
The invention relates to recombinant gene sequences for synthesizing a protein having the biological activity of the PilC protein. Furthermore, the invention relates to DNA recombinant methods for the production of proteins having the biological activity of the PilC protein as well as the molecular-biological tools required therefor. In addition, the invention relates to proteins having the biological activity of the PilC protein and its antibodies. Other embodiments of the invention are pharmaceutical compositions comprising the mentioned proteins or antibodies. Preferably, these pharmaceutical compositions serve as vaccines for the immunization against pathogenic bacteria bearing type 4 pili. The invention also relates to kits for the detection of bacteria bearing type 4 pili or antibodies directed against them comprising the mentioned proteins or antibodies. Finally, the invention relates to cellular receptors for bacteria bearing type 4 pili and analogues thereof.
A crucial step in the occurrence and the manifestation of any infection is the attachment of the pathogen to certain molecular structures (receptors) of the host organism. The pathogen's structures responsible for attachment shall be referred to as adhesins. It is possible that multiple molecular interactions between adhesins of the pathogen and receptors of the host organism are necessary for the occurrence and/or the manifestation of an infection. On the other hand, it is thinkable that blocking one single molecular interaction between an important adhesin and a receptor is sufficient to prevent an infection. Blocking molecular adhesin-receptor interactions is a possible form of prevention and/or therapy of infectious conditions. Prophylactic approaches include, for instance, the formation of antibodies directed specifically against the adhesin and block any interaction with its receptor by active immunization (vaccination). However, the interaction may principally also be prevented in the sense of a prophylactic or therapeutic measure by passively administered antibodies or other substances, for instance adhesin or receptor analogous substances, which shall be referred to under the generic term inhibitors. Important adhesins of numerous gram negative pathogens are pili (also termed fimbriae or fibrillae). They are polymeric structures forming fine thread-like appendages to the surface of the bacteria. Pili make it possible for the bacteria to attach to specific receptors of the host organism via the adhesins contained in the pili. In some cases it was shown that the loss of the pili leads to the pathogen's loss of infectiousness. This loss of infectiousness can be explained by the loss of the capability to form attachments. Therefore, blocking the molecular interaction between pilus adhesin and receptor can avoid or stop the infection of the host organism.
In gram negative bacteria, different types of pili are known. The majority of all known pili are heteropolymeric structures comprising several subunits, one main subunit and other inferior subunits which are present in only a few copies. In some well examined cases the inferior subunits are the actual adhesive structures (adhesins), whereas the main subunit which is present in a high number of copies takes over the function of a framework (Lindberg et al., Nature 328, 84-87, 1987). Numerous pathogenic gram negative bacteria species form type 4 pili, which are also referred to as N-Me-Phe or type IV pili. They include the pathogenic Neisseria species
N. gonorrhoeae
and
N. meningitidis,
causing gonorrhoe and bacterial meningitis, respectively, in humans. However, there is no effective vaccine against
N. gonorrhoeae
so far. The capsula-specific vaccines against some sero groups of
N. meningitidis,
however, are available; unfortunately they offer only partial protection and are considered immunologically problematical. Thus, these infections are usually treated with antibiotics; however, the ever increasing resistance of the pathogens to antibiotics causes problems. The development of alternative methods of treatment is therefore urgent. Another important pathogen forming type 4 pili in humans is
Pseudomonas aeruginosa
(Sastry et al., FEBS Letters 151, 253-255, 1983), which is a central problem for patients suffering from cystic fibrosis, immunodeficiencies and in connection with septic infections. Effective vaccines and/or inhibitors of this pathogen are not yet available; problematic is above all the increased spreading of multi-resistant strains. Alternative methods of treatment are thus urgently required in this field too. More examples of important pathogens forming type 4 pili are enteropathogenic
Escherichia coli
(EPEC; Giron et al., Science 254, 710-713, 1991),
Vibrio cholerae
(Shaw et al., Infect. Immun. 58, 3042-3049, 1990),
Bacteroides nodosus
(McKern et al., FEBS Letters 164, 149-153, 1983),
Moraxella bovis
(Marrs et al., J. Bacteriol. 163, 132-139, 1985) and other pathogens, causing diseases in humans and animals and vaccines against which are searched for intensively. Type 4 pili are defined by the structure of their main subunit, usually referred to as pilin; besides, there are specific terms for the pilin main subunit for individual species of bacteria, e.g. PilE for
N. gonorrhoeae
or PilA for
Pseudomonas aeruginosa.
The pilin's structure of the type 4 pili differs substantially from that of main subunits of other pilus types, such as the group of pap-like pili (Baga et al. J. Bacteriol. 157, 330-333, 1984). Characteristics of the pilin of type 4 pili are (a) the short, positively charged, aminoterminal signal sequence of the pilin's preform (exception
V. cholerae
type 4 pilin), (b) the hydrophobic amino terminal region of the mature pilin that exhibits strong sequence homology between different type 4 pilins, (c) the modification of the amino terminal Phe residue of the mature pilin by means of a methyl group in N position (N-Me-Phe), (d) two Cys residues in the carboxy terminal region of the pilin that form a loop and (e) a property termed twitching motility.
So far, PilC proteins were detected as components of N. gonorrhoeae and
N. meningitidis.
Up to now, an assembly function in the pilus biogenesis was considered to be a function of these PilC proteins (Jonsson, Dissertation, New Series No. 322, University of Umea, ISSN 0346-6612). However, these works never indicated a possible role as an important adhesin. In other tests, scientists succeeded in isolating mutants of
N. gonorrhoeae
which did still form pili but no longer exhibited an adherence to epithelial cells (Rudel et al., Mol. Microbiol. 6, 3439-3450, 1992). These mutants turned out to be phase-variant strains that had stopped forming PilC proteins. A direct function of the Neisseria PilC proteins could not be derived from these experiments. However, the experiments showed that the assembly of the Neisseria pili can take place also if no PilC proteins are present. Scientists had been successful in cloning a single PilC-protein encoding gene in
E. coli.
This was achieved by means of the gel electrophoretic purification of PilC protein from isolated Neisseria pili (Jonsson et al., EMBO J. 10, 477-488, 1991). The gel electrophoretically purified PilC protein was used for the recovery of antiserum. The gel electrophoretically purified PilC protein had no biological activity in the sense of this invention. The corresponding antiserum thus does not have the property of this invention to block the attachment of piliated Neisseria to epithelial cells. Using the antiserum, scientists at first succeeded in identifying an
E. coli
phage clone carrying a partial PilC-encoding gene. An
E. coli
clone with an intact PilC-encoding gene could be identified by means of the partial PilC-encoding gene using DNA hybridization (Jonsson et al. EMBO J. 10, 477-488, 1991). This recombinant PilC-encoding gene
Meyer Thomas Franz Ferdinand
Rudel Thomas
Ryll Roland Richard
Scheuerpflug Ina Bärbel
Birch & Stewart Kolasch & Birch, LLP
Longton Enrique D.
Max-Planck-Gesellschaft zur Forderung der Wissenschaften e.v.
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