Drug – bio-affecting and body treating compositions – Whole live micro-organism – cell – or virus containing – Genetically modified micro-organism – cell – or virus
Reexamination Certificate
1997-05-30
2004-08-03
Devi, S. (Department: 1645)
Drug, bio-affecting and body treating compositions
Whole live micro-organism, cell, or virus containing
Genetically modified micro-organism, cell, or virus
C424S200100, C424S201100, C424S203100, C424S184100, C424S093400, C424S236100, C424S235100, C424S234100, C435S245000, C435S069300, C435S071100, C435S252300
Reexamination Certificate
active
06770275
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to live attenuated RTX-producing bacteria of the family Pasteurellaceae, methods for the production of such bacteria, to vaccines comprising such bacteria, methods for the production of such vaccines and methods for the protection of man and animals against infection with virulent RTX-producing bacteria of the family Pasteurellaceae.
BACKGROUND OF THE INVENTION
The family of Pasteurellaceae comprises the genera Haemophilus, Actinobacillus and Pasteurella. The bacteria of this family are also often referred to as bacteria of the HAP-group. Several species of these closely related genera are known to express homologous calcium-dependent, pore-forming cytotoxins, the so-called RTX toxins. (RTX stands for repeat in toxin). RTX toxin-producing bacteria of this family are the cause of a whole range of infectious diseases, influencing both man and animals.
RTX toxins are also known from other genera, not related to the HAP-group, such as Escherichia and Bordetella. These RTX toxins in some respects resemble the RTX-toxins of the HAP-group.
The RTX toxins have been extensively reviewed by Braun et al. (Critical Rev. in Microbiol. 18(2): 115-158 (1991) Gram-negative strains have also been reviewed in Welch, R. A. (Molecular Microbiology 5/3: 521-528 (1991)) and in Welch et al. (Inf. Agents and Disease 4: 254-272 (1995)).
It is the presence of the RTX toxins in the RTX-producing members of the Pasteurellaceae family of bacteria, that highly contributes to the pathogenic character of these bacteria for both man and animals.
All RTX toxins display some kind of cytotoxic or cytolytic activity. The target-cell- and host-specificity differ however, depending on the toxin and on differences in acylation (McWhinney et al.; J. Bact. 174: 291-297 (1992) and Hackett et al.; J. Biol. Chem. 270: 20250-20253 (1995)). As a result of the difference in target cells, the various toxins of the RTX toxin family are known e.g. as haemolysin, cytolysin or cytotoxin.
Although many RTX-producing members of the HAP-group are known, some of them are notorious for the economic damage they cause.
Actinobacillus pleuropneumoniae
produces RTX toxins that are cytotoxic/cytolytic to pig, horse, bovine and human erythrocytes, to rabbit and porcine neutrophils and to porcine alveolar macrophages. (Rosendal et al; Am. J. Vet. Res. 49: 1053-1058 (1988), Maudsley J. R. and Kadis S; Can. J. Microbiol. 32: 801-805 (1986), Frey. J and Nicolet. J; Inf. & Imm. 56:2570-2575 (1988), Bendixon et al; Inf. & Imm. 33: 673-676 (1981), Kamp, E. M. and van Leengoed, L. A. M. G.; J. Clin. Microbiol. 27: 1187-1191 (1989)).
Actinobacillus infection in pigs causes severe economic losses to pig industry, due to acute mortality in young pigs and reduced weight gain in older animals.
The
Pasteurella haemolytica
RTX toxin activity is mainly directed against neutrophils and monocytes/macrophages from ruminants (Shewen and Wilie; Inf. & Immun. 35, 91-94 (1982), Baluyut et al.; Am. J. Vet. Res. 42: 1920-1926 (1981), Himmel et al.; Am. J. Vet. Res. 43: 764-767 (1982)).
Pasteurella infections cause severe problems in ruminants, especially cattle and sheep.
Mastitis and pneumonia are seen in both sheep and cattle, whereas Shipping Fever causes additional problems in cattle. Economic losses due to Pasteurella infections are high. Other, non-HAP-group bacteria are also known to produce RTX toxins.
The
E. coli
haemolysin is toxic for a large variety of cells, from a large number of different animal species. It lyses erythrocytes from many animal species within a few minutes after contact. (Cavalieri, S. J. and Snyder, I. S.; Inf. & Imm. 37: 966-974 (1982), Gadeberg et al; Inf. & Imm. 41: 358-364 (1983), Keane et al; Am. J. Pathol. 126:305-357 (1987), Bhadki et al; J. Exp. Med. 169: 737-754 (1989)).
The
Bordetella pertussis
haemolysin also displays a large host-cell range. (Shattuck, R. L. and Storm, D. R.; Biochemistry 24: 6323-6328 (1985), Hewlett et al, In Protein Bacterial Toxins, Rappuoli, R. et al. (Eds), Stuttgart, Fisher-Verlag 249-257 (1990)).
The genetic organisation of the operons involved in the synthesis, activation and transportation of the RTX toxins in Gram-negative bacteria has been reviewed recently by Coote, J. G. (FEMS Microbiology reviews 88: 137-162 (1992)) In general, the RTX operon contains four genes: the actual Toxin gene (A), an Activator gene (C), and two genes (B and D (and E in
Bordetella pertussis
)) encoding proteins involved in secretion of the toxin into the surrounding medium. The primary translation product of the Toxin-gene (A) is a non-toxic protein.
The role of the Activator gene (C) is of paramount importance in that the gene product encoded by this gene activates the toxic activity of the RTX toxin by posttranslational modification.
This activation results in a structural modification of the toxin. In e.g.
Bordetella pertussis,
the posttranslational no modification of the RTX toxin is caused by amide-linked palmitoylation of a lysine residue (Hackett et al.; Science 266: 433-435 (1994). The RTX toxin of
E. coli
could be activated in vitro by transfer of a fatty acyl group from acyl carrier protein to prohaemolysin (Issartel et al.; Nature 351: 759-761 (1991)).
It is known (see e.g. Coote, J. G.; FEMS Microbiology reviews 88: 137-162 (1992)), that RTX toxins are important virulence factors in bacteria belonging to the Pasteurellaceae. This has been shown for e.g.
Actinobacillus pleuropneumoniae
by Tascon et al.(Mol. Microbiol. 14: 207-216 (1994)) and by Jansen et al. (Inf. & Imm. 63: 27-37 (1995)).
Virulence factors are known to be the main targets for incorporation in vaccines.
Therefore, several attempts have been made to use RTX toxins as subunit vaccines.
In vivo synthesised RTX toxins of the HAP-group are per se produced in the presence of the RTX Activator protein. Therefore, RTX toxins are always posttranslationally modified into highly toxic proteins.
Given their high toxicity it is clear that the RTX toxins need to be detoxified before they can be used as a vaccine component.
Subunit vaccines based on in vivo synthesised RTX toxins from
A. pleuropneumoniae
that lost their toxicity have been described earlier, e.g. in European Patent EP No. 0.354.628, in which subunit vaccines based upon a haemolysin and a cytotoxin of
A. pleuropneumoniae
are disclosed, and in European Patent EP No 0.453.024, in which
A. pleuropneumoniae
subunit vaccines based upon haemolysins, cytotoxins and outer membrane proteins are disclosed.
Subunit vaccines based on RTX toxins from
Pasteurella haemolytica
have also been disclosed, e.g. in U.S. Pat. No. 5,055,400, Canadian Pat. Appl. CA 2,014,033 and Canadian Pat. Appl. CA 2,081,950.
RTX toxins as subunits for use in vaccines are easily obtained from the supernatant of bacterial cultures of the wild-type strains. Another way of obtaining the RTX toxin as a subunit has been proposed in Canadian Patent Application CA 2,045,950, in which heterologous expression of the genes encoding the
A. pleuropneumoniae
RTX-protein in the heterologous bacterial strain
E. coli
has been described. No vaccine experiments with the RTX toxins so obtained were shown however.
A comparable approach for the production of subunit vaccines has been proposed in European Patent EP 0.500.736. In this patent, the sequence of the RTX Toxin gene (A) and an Activator gene (C), is disclosed. Also a heterologous expression system for the expression of the Toxin gene A in the presence or absence of the Activator gene C is disclosed. No vaccination experiments with the toxin subunit were however, described.
There are however, three important disadvantages to all RTX toxin subunit vaccines:
high amounts of antigenic material are needed in order to adequately trigger the immune system.
usually, only B-cell immunity is triggered.
a live pathogenic bacterium has many important immunogenic molecules, such as Outer Membrane Proteins and capsular polysaccharides, all being important for protection. Therefore, in order to produce an efficient subunit
Frey Joachim
Segers Ruud Philip Antoon Maria
Van Den Bosch Johannes Franciscus
Akzo Nobel N.V.
Blackstone William M.
Devi S.
Ramey III William P.
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