Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Bacterium or component thereof or substance produced by said...
Reexamination Certificate
1998-06-23
2003-08-26
Devi, S. (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, C424S200100, C424S201100, C424S236100, C424S203100, C424S184100, C424S093400, C424S093200, C435S069300
Reexamination Certificate
active
06610307
ABSTRACT:
The present invention relates to modified microorganisms suitable for use as live vaccines. The present invention also relates to the use of modified microorganisms as biological vectors. The present invention further relates to vaccine compositions.
Bovine respiratory disease (BRD) complex, shipping fever, or pneumonic pasteurellosis, is a multifactorial disease whereby a combination of viral infection, adverse environment and poor immune status may combine to predispose animals to bacterial infections. BRD is a major cause of economic loss in the cattle feed lot industry. The principal microorganism associated with the disease is the bacteria
Pasteurella haemolytica
serotype 1. (Schiefer, et al., 1978). Under normal conditions
P. haemolytica
is a component of the normal flora of the upper respiratory tract, it is only when pulmonary clearance mechanisms are impaired that colonisation of the lung occurs resulting in disease (Frank and Smith, 1983). A number of virulence factors have been associated with
P. haemolytica
, including surface structures such as the capsular polysaccharide (Adam et al. 1984), and a secreted exotoxin which is heat labile and specific for ruminant leukocytes (Shewen and Wilkie, 1982).
The exotoxin, or leukotoxin (Lkt), may contribute to pathogenesis by impairing the primary lung defenses and subsequent immune responses or by causing inflammation as a result of leukocyte lysis. Characterisation of the Lkt has shown it to be a member of the RTX family of toxins (Strathdee and Lo, 1989) which are produced by a variety of bacteria including Actinobacillus spp,
Proteus vulgaris, Morganella morganii, Bordetella pertussis
, and the most characterised produced by
E. coli
. All RTX toxins function by producing pores in the target cells, thereby interrupting osmotic balance, leading to rupture of the target cell. Although the mode of action is identical for RTX toxins their target cells vary greatly in type and cross-species specificity. Structurally, this family of toxins are characterised by the presence of glycine rich repeat structures within the toxin that bind to calcium and may have a role in target cell recognition and binding, a region of hydrophobic domains that are involved in pore formation, the requirement for post translational activation, and dependence on a C-terminal signal sequence for secretion. Production and secretion of an active RTX toxin requires the activity of at least four genes, C, A, B, and D. The A gene encodes the structural toxin, the C gene encodes a post-translational activator and the B and D genes encode proteins that are required for secretion of the active toxin. The Lkt is encoded by an operon that consists of the four contiguous genes (CABD), transcribed by a single promoter. The Lkt differs from a number of other RTX toxins, which have a broad host cell specificity, by having a target cell specificity restricted to ruminant leukocytes (Reviewed: Coote, 1992).
The Lkt has also been associated with protective immunity; with anti-toxin antibodies in the field relating to disease resistance, and a commercial culture supernatant vaccine (Presponse; Langford Inc., Guelph, Ontario, Canada) containing Lkt showing efficacy in reducing the incidence and severity of pneumonia following experimental challenge and in the feed lot (Gentry et al., 1985; Mosier et al., 1986; 1989; Shewen and wlkie, 1987; Shewen et al., 1988). This culture supernatant vaccine, in addition to inducing anti-Lkt antibodies, also stimulates an immune response to other soluble antigens present in the culture supernatant, and therefore a direct correlation between anti-Lkt and protection can not be claimed.
The use of
Pasteurella bacterins
(inactive vaccines) in the field has had limited success in controlling pneumonic pasteurellosis, in several field trials the administration of bacterin based vaccine has not protected against disease or in some cases had led to an enhancement of disease (Bennett, 1982; Morter et al., 1982). Bacterin vaccines also have the disadvantages of requiring the use of adjuvants, may result in site reactions, and in a number of cases require multiple dose to obtain protection.
It is an object of the present invention to alleviate one or more of the problems of the prior art.
Accordingly, in one aspect the present invention provides a modified microorganism which produces an Lkt toxin, wherein said Lkt toxin is partially or fully inactivated.
The term “modified” includes modification by recombinant DNA techniques or other techniques such as chemical- or radiation-induced mutagenesis. Where recombinant DNA techniques involve the introduction of foreign DNA into host cells, the DNA may be introduced by any suitable method. Suitable methods include transformation of competent cells, transduction, conjugation and electroporation.
In a further embodiment of the present invention, there is provided a modified microorganism wherein an Lkt toxin operon including an Lkt structural gene and/or a post translational activator of the organism is partially or fully inactivated.
The term “Lkt toxin operon” as used herein the claims and description is intended to include those genes involved in the expression of an Lkt toxin being a product of the Lkt toxin operon. The genes included in the Lkt toxin operon include the post translational activator gene (C), the structural gene (A), and the B and D genes which encode proteins that are required for secretion of the activated Lkt toxin.
The term “partially or fully inactivated” as used herein the claims and description includes modification of a gene by recombinant DNA techniques including introduction and deletion of DNA from the gene including single or multiple nucleotide substitution, addition and/or deletion including full or partial deletion of the gene, using a target construct or plasmid segregation; and
chemical induced-, radiation induced- or site specific mutagenesis.
The present applicants have found that a precursor of Lkt toxin has reduced toxic activity. Surprisingly, the present applicants have also found that the Lkt toxin precursor is capable of inducing an immune response in an animal that offers cross protection against heterologous challenge with a microorganism which produces the Lkt toxin.
Accordingly, in a preferred embodiment of the invention the inactivated Lkt toxin is a precursor of Lkt toxin. The precursor may be an unprocessed expression product of the Lkt structural gene. The Lkt structural gene may be the Lkt A gene.
The microorganism may be one which does not naturally produce an Lkt toxin. The microorganism may be a bacterium, virus or fungus into which the Lkt structural gene, such as the Lkt A gene, has been introduced.
In a preferred embodiment, however, the microorganism is one which naturally produces an Lkt toxin. The microorganism which naturally produces an Lkt toxin may be
Pasteurella haemolytica.
The present applicants have found that a microorganism which naturally produces an Lkt toxin may be engineered to produce an inactive Lkt toxin precursor by eliminating the post-translational activator of the precursor product. Accordingly, in a preferred embodiment the microorganism is unable to produce a post-translational activator of the Lkt toxin precursor or produces an inactivated post-translational activator of the Lkt toxin precursor. The post-translational activator may be a product of the Lkt C gene.
In a preferred embodiment the Lkt C gene of the microorganism is inactivated or partially or fully deleted. The Lkt C gene may be inactivated by site specific mutagenesis. The Lkt C gene may be inactivated by any single or multiple nucleotide substitution, addition and/or deletion. Preferably, the Lkt C gene is inactivated by homologous recombination using a targeting construct. The targeting construct may include a selectable marker flanked by sequences homologous to sequences flanking the desired insertion site. The selectable marker may be a gene which confers resistance to a toxic substance such as mercury or may be an antibiotic resistance det
Hodgson Adrian Leslie Mark
Prideaux Christopher Thomas
Devi S.
Jacobson & Holman PLLC
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