Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Using a micro-organism to make a protein or polypeptide
Reexamination Certificate
1998-06-19
2002-10-29
Graser, Jennifer E. (Department: 1645)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Using a micro-organism to make a protein or polypeptide
C435S320100, C435S173300, C435S173300, C435S243000, C435S252300, C435S252800, C435S069100, C435S069300, C435S069700, C536S023100, C536S023700, C424S184100, C424S234100, C424S236100, C424S235100
Reexamination Certificate
active
06472183
ABSTRACT:
The present invention relates to modified microorganisms suitable for use as live vaccines. The present invention also relates generally to the use of modified microorganisms as biological vectors. The present invention further relates to vaccine compositions. In particular, the present invention relates to compositions suitable for inducing an immune response against RTX toxins.
Actinobacillus pleuropneumoniae
(APP), is a member of the family Pasteurellaceae, and is the aetiological agent of porcine pleuropneumonia, an acute or chronic infection of pigs characterised by haemorrhagic, fibrinous and necrotic lung lesions (Pohl et. al., 1983; Kilian and Biberstein, 1984). The disease is highly contagious, and associated with all ages of growing pigs, resulting in severe economic losses to the swine industry. The direct mode of transmission of APP means that infection is more prevalent under intensive breeding conditions, fortunately the host range of APP is restricted to pigs reducing the potential sources of infection. To date, twelve serovars of APP have been identified worldwide (Serovars 1-12) with Serovars 1, 7 and 12 making up approximately 90% of Australian isolates (Kamp and Shope, 1964). A number of potential virulence factors have been identified including outer membrane proteins (Mulks and Thacker, 1988; Rapp and Ross, 1988), lipopolysaccharide (Udeze et al., 1987; Fenwick and Osburn, 1986), capsule (Inzana et al., 1988; Rosendale and Macinnes, 1990; Lenser et. al., 1988) and secreted toxins (Rycroft et al., 1991; Bhatia et al., 1991; Fedorka-Crey et al., 1990). The secreted toxins, or APX toxins, are members of the RTX toxin family (Frey et al., 1993, 1994).
RTX toxins are produced by a number of gram negative bacteria including Actinobacillus spp,
Proteus vulgaris, Morganella morganii, Bordetella pertussis, Pasteurella haemolytica
, and the most characterised of the group produced by
E. coli
(Welch, 1991) . 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 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 (Reviewed: Coote, 1992).
At least three different APX toxins are produced by APP, designated APX1, APX2, and APX3. APX1 shows strong, and APX2 relatively low, haemolytic activity, both are cytotoxic and active against a broad range of cells of differing types and species (Frey and Nicolet, 1988; Rosendale et al., 1988; Kamp et al, 1991). APX3 is nonhaemolytic, but is strongly cytotoxic, with a host range including porcine alveolar macrophages and neutrophils (Rycroft et al., 1991; Kamp et al., 1991). No serovar of APP produces all three APX toxins, the majority produce two (APX1 and 2: Serovars 1, 5, 9, and 1 1; APX2 and 3: 2, 3, 4, 6, and 11) with a small number (APX1: 10, APX2: 7 and 12) producing only one APX (Frey and Nicolet, 1990; Frey et al., 1992,1993,1994; Kamp et al., 1991; Rycroft et al., 1991) The pattern of APX production appears to be associated with virulence, with those serovars producing APX1 and 2 being the most virulent (Frey et al., 1994; Komal and Mittal, 1990). Production and secretion of active RTX toxins requires the activity of at least four genes, C, A, B, and D. The A gene encodes the structural toxin, the C gene encodes the post-translational activator and the B and D genes encode proteins that are required for secretion of the activated toxin (Issartel et al., 1991; Welch, 1991; Felmlee et al., 1985). APX1 and 3 are encoded by operons that consist of the four contiguous genes (CABD), whilst the APX2 operon contains only the C and A genes, and in some cases remnants of the B gene. Secretion of APX2 is dependent on the activity of the APX1B and D gene products (Reviewed Frey et al., 1994).
Virulence analysis of spontaneous, and chemically induced, non-haemolytic mutants indicated a role of the APX toxins in virulence, which has recently been confirmed using transposon mutagenesis (Anderson et al., 1991; Gerlach et al., 1992; Inzana et al., 1991; Rycroft et al., 1991a,b; Tacon et al., 1993, 1994). Complete protection from disease and/ or carrier status cannot be obtained using vaccines comprising chemically inactivated bacteria, or purified subunit vaccines comprising outer membrane proteins, lipopolysaccharides, or capsule. In comparison complete protection from disease in mice was obtained following vaccination with purified APX combined with formalised whole cells, indicating a role for the APX toxins in protective immunity (Bhatia etal., 1991).
Vaccination against pleuropneumonia, resulting from APP infection of pigs, has utilised, to date, bacterins or subunit vaccines based on various components of the bacteria. Results obtained with inactive vaccines have offered, at best, homologous protection against the serovar used to prepare the vaccine material. Currently twelve known serovars of APP exist, of varying virulence, each requiring a different vaccine preparation. To date commercial vaccines have been formulated to contain a number of serovars, offering protection against the most frequently observed serovars in that geographic location. In contrast to inactive vaccines, natural infection with any one serovar offers protection against reinfection with any other serovar, indicating the potential of a live vaccine to offer cross protection against APP serovars.
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 RTX toxin, wherein said RTX 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 RTX toxin gene including an RTX structural gene and/or a post translational activator of the organism is partially or fully inactivated.
The term “RTX toxin gene” as used herein the claims and description is intended to include those genes involved in the expression of an RTX toxin being a product of the RTX toxin gene. The genes included in the RTX toxin gene 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 RTX 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 an RTX toxin has reduced toxic activity. Surprisingly, the present applicants have also found that the RTX 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 RTX toxin.
Accordingly, in a preferred embodiment of the invention the inactivated RTX toxin is a precursor of an RTX toxin. The precursor may be an unprocessed expression product of
Hodgson Adrian Leslie Mark
Prideaux Christopher Thomas
Commonwealth Scientific and Industrial Research Organisation
Graser Jennifer E.
Jacobson & Holman PLLC
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