Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Bacterium or component thereof or substance produced by said...
Reexamination Certificate
1999-08-31
2004-08-31
Smith, Lynette R. F. (Department: 1645)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Bacterium or component thereof or substance produced by said...
C424S009200, C424S184100, C424S192100, C424S193100, C424S197110, C424S234100, C424S236100, C424S278100, C435S220000, C435S235100, C435S340000, C930S200000
Reexamination Certificate
active
06783764
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to live attenuated bacteria of the genus
Actinobacillus pleuropneumoniae,
having a mutation in a gene encoding a toxin, methods for the production of such bacteria, to vaccines comprising such bacteria, methods for the production of such vaccines, to vaccines comprising a toxin, methods for the production of such vaccines and methods for the protection of animals against infection with bacteria of the genus,
Actinobacillus pleuropneumoniae.
BACKGROUND OF THE INVENTION
Bacteria belonging to the genus Actinobacillus all produce so-called RTX-toxins. (RTX stands for repeat in toxin).
It is the presence of these RTX-toxins that highly contributes to the pathogenic character of these bacteria.
The RTX-toxins have been extensively reviewed by Braun et al. (Critical Rev. in Microbiol. 18(2): 115-158 (1991)). RTX-toxins in 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)).
All known 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. The genus Actinobacillus comprises a number of different species, inter alia,
Actinobacillus pleuropneumoniae, A. actinomycetemcomitans, A. suis, A. rossii, A. equuli
and
A. lignieresii.
Actinobacillus pleuropneumoniae
produces serotype-dependent 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)).
Infections with Actinobacillus in pigs are the cause of severe economic losses to pig industry, due to acute mortality in young pigs and reduced weight gain in older animals.
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)). Frey has specifically reviewed the known three RTX-toxins in
Actinobacillus pleuropneumoniae
in Bacterial Protein Toxins, Zbl Bakt. Suppl. 24, p. 322-, Freer et al. (Eds.), Gustaf Fischer, Stutttgart, Jena, New York, 1994.
In
Actinobacillus pleuropneumoniae,
this kind of RTX-operon contains four genes: the actual Toxin-gene (A), an Activator-gene (C), and two genes (B and D) 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, of which the toxic activity is activated by the Activator-gene (C) product.
Until recently, it was assumed that only three RTX-toxins, all having the above-described genetic organisation or at least having the Toxin-gene (A) and Activator-gene (C), existed in Actinobacillus species.
These three RTX-toxins, ApxI, Apx-II and Apx-III have respectively a pronounced haemolytic activity (ApxI), a mild haemolytic activity (Apx-II) or a macrophage-cytotoxic activity (Apx-III).
The various toxic activities are fairly randomly divided over the serotypes. There are four subgroups:
a subgroup A, represented by serotypes 1, 5, 9 and 11, producing ApxI and Apx-II,
a subgroup B, represented by serotypes 2, 3, 4, 6 and 8, producing Apx-II and Apx-III,
a subgroup C, represented by serotype 10, producing ApxI only,
a subgroup D, represented by serotype 7 and 12, producing Apx-II only,
It is known, that ApxI, -II, and -III all are essential elements in universal vaccines against
Actinobacillus pleuropneumoniae
infection: a vaccine not comprising at least ApxI, -II, and -III will not provide protection against all
Actinobacillus pleuropneumoniae
serotypes. Also, a vaccine not comprising at least the Apx-toxins of one specific serotype will not even induce protection against that single serotype.
Subunit vaccines based on in vitro 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.
There are however four important disadvantages to subunit vaccines in general:
high amounts of antigenic material are needed in order to adequately trigger the immune system.
usually, only B-cell immunity is triggered.
several protective antigens are only triggered in vivo, and therefore can not be present in subunit vaccines.
a live pathogenic bacterium has many important immunogenic molecules, such as Outer Membrane Proteins and capsular polysaccharides, all potentially important for protection and thus to be included in an efficient subunit vaccine.
Next to the obvious problems mentioned under points one and two, especially the fourth point makes it difficult to make an efficient subunit vaccine.
This is e.g. illustrated by the
A. pleuropneumoniae
subunit vaccine disclosed in European Patent EP No 0,453,024 mentioned above, in which four different subunits (three RTX-toxins and an outer membrane protein) are combined in one vaccine.
It is clear, that in order to overcome the disadvantages of subunit vaccines against Pasteurellacea-infection, a live attenuated vaccine would be highly desirable.
A live attenuated vaccine has the following advantages:
it can be administered in low doses (it is self-replicating)
it closely mimics the natural/wild-type infection
it provides all the possible immunologically important antigens at the same time.
Nevertheless, in spite of the clear advantages, no live vaccines based on
Actinobacillus pleuropneumoniae
were commercially available prior to the present invention.
The reason for this lies in the following paradox: as mentioned before, ApxI, -II, and -III all are essential elements of universal vaccines against
Actinobacillus pleuropneumoniae
infection. Live vaccines therefore have to produce these three RTX-toxins. These three RTX-toxins are however strong virulence factors in all Actinobacillus species (see e.g. Coote, J. G.; FEMS Microbiology reviews 88: 137-162 (1992), Tascon et al.; Mol. Microbiol. 14: 207-216 (1994)), Jansen et al.; Inf. & Imm. 63: 27-37 (1995)).
Deletion of the RTX-toxins in order to attenuate the virulence of live App strains is technically feasible, but this does not provide a solution for the dilemma: such RTX-negative strains would be useless as live attenuated vaccine strains since they do no longer induce immunity in the host against the haemolytic/cytotoxic activity of
Actinobacillus pleuropneumoniae
field strains.
Virulence factors that, although important in the induction of immunity, do play a less important role in building up immunity than ApxI, -II and -III, and thus can in principle be deleted are however currently not known.
It would thus be highly desirable to have a site on the genome of App that attributes to virulence and therefore leads to an attenuated App strain when modified, whereas at the same time it is, although useful in triggering immunity, dispensable from a vaccine point of view. No such sites are however currently known. Moreover, it would be highly desirable if such a site would be universally present in all App strains, instead of being restricted to certain serotypes. Such a site would then allow all different serotypes to be attenuated by deletion of
Frey Joachim
Segers Ruud Philip Antoon Maria
Akzo Nobel NV
Hines Ja'Na
Raine, III William P.
Smith Lynette R. F.
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