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
1996-03-22
2001-11-27
Graser, Jennifer E. (Department: 1645)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C536S023700, C435S069100, C435S243000, C435S252300, C435S320100
Reexamination Certificate
active
06323005
ABSTRACT:
1. FIELD OF THE INVENTION
The present invention relates to the gene encoding the transferrin-binding protein 1 (thereafter “Tbp1”) from
Actinobacillus pleuropneumoniae
. The invention also relates to recombinant plasmids comprising the gene, expression systems for the gene, the use of either the expressed Tbp1 or its antigenic fragments for the formulation of vaccines against porcine pleuropneumonia. Either Tbp1 or fragments thereof can be used with other purposes, such as eliciting antibodies and preparation of diagnostic reagents to disease.
2. BACKGROUND OF THE INVENTION
Porcine Pleuropneumonia
Actinobacillus pleuropneumoniae
(thereafter “App”) is a Gram-negative bacteria which causes porcine pleuropneumonia, a world-wide distributed infectious disease responsible for great economic losses in the swine industry (Nicolet, J., 1992. In: Leman, A. D., Straw, B., Mengeling, W. L., D'Allaire, S., Taylor, D. J., Eds.
Diseases of swine. Ames
, Iowa State University Press, pp. 401-408).
On the basis of &bgr;-nicotine-adenine dinucleotide requirements (thereafter “NAD”), App can be divided into biotype 1, NAD-dependent strains and biotype 2, NAD-independent strains. So far, 12 serotypes have been described in biotype 1 being serotype 5 often subdivided into 5
a
and 5
b
(Nielsen, R., 1986
. Acta Vet Scand
. 27, 453-455), and 2 serotypes in biotype 2 (Fodor, L., Varga, J., Molnar, E. and Hajtos, I., 1989
. Vet. Microbiol
. 20: 173-180). Serotyping is mainly based on capsular antigens.
The virulence of the agent seems to be more or less related to the serotype, although all App serotypes described in both biotypes may cause the disease and death in pigs. Biotype 2 strains show less virulence than those of biotype 1, and field observations indicate that serotypes 1, 5
a,
5
b,
9 and 10 are more virulent than the other biotype 1 serotypes (Dom, P. and Haesebrouck, F., 1992
. J. Vet. Med. B
., 39: 303-306).
The main route of App spread is airborne and the disease is transmitted mainly by direct contact from pig to pig or by droplets within short distances. In acute outbreaks the infection may jump from one pen to another, suggesting the possible role of aerosols in connection with long distances or the indirect transmission of contaminated exudate by farm personnel. It is generally accepted that App enters the pulmonary alveoli directly via the trachea and bronchi. The clinical signs of porcine pleuropneumonia vary with the state of immunity of the animals, the environmental stress or the degree of exposure to the infectious agent. All age categories may suffer the disease, but young fattening pigs are more frequently affected. The clinical course may be peracute, acute or chronic. In the peracute form, there is an intense inflammatory response, with haemorrhage, edema and fibrinous exudation. The acute form of the disease is characterized by extensive haemorrhage and fibrinous exudation in the pulmonary parenchyma and the pleural cavity, being defined as fibrinous pleuritis and haemorrhagic necrotizing pneumonia that causes the death of the animal within a period of 24-48 hours. Pigs that survive the disease may become carriers of the pathogen, and develop chronic injuries as pulmonary necrosis, nodules and fibrinous adherences in the pleural cavity. Other signs are lack of appetite and decreased gain in body weight (Nicolet, 1992).
Economic losses due to acute outbreaks of the disease result mainly from the high mortality (1-10% or more) and costs for medication (Desrosiers, R., 1986. Vet. Rec
.
119
:
89
-
90
). Economic losses resulting from chronic cases of pleuropneumonia are due to a decreased growth rate of the affected animals.
Virulence Factors or App
Capsule
All App strains possess an oligosaccharide capsule that protects the agent from phagocytosis and complement lysis. This structure seems to contribute to the agent virulence and may play a role in serotype-specific partial protection induced by vaccination with bacterins.
Lipopolysaccharides
Lipopolysaccharides (thereafter “LPSs”) are the main constituents of the outer membrane of Gram-negative bacteria. They consist of a polysaccharide and a lipid A moiety, the latter being an endotoxin. Purified App LPSs induce non haemorrhagic nor necrotic lesions in pig lungs, which indicate that LPSs are not responsible for the typical App lesions, but may contribute to their formation (Jansen, R., 1994. PhD Thesis, Utrecht, The Nederlands).
Outer-membrane Proteins
Convalescent animal sera recognize several proteins from the outer membrane of App (thereafter “OMPs”). Some of them, specifically induced under iron restriction, will be discussed further. OMP electrophoretic profiles differ for most serotypes of App (Rapp, V. J., Munson, R. S. and Ross, R. F., 1986
. Infect. Imm
. 52: 414-420), although three proteins of 17, 32 and 42 kilodaltons (thereafter “kDa”) are immunologically dominant (Macinnes, J. I. and Rosendal, S. 1987
. Infect. Imm
. 55: 1626-1634). Immunization with a crude extract of outer membranes conferred a limited protection to the challenge with the pathogen (Beaudet, R., McSween, G., Boulay, G., Rousseau, P., Bisaillon, J. G., Descoteaux, J. P. and Ruppaner, R. 1994
. Vet. Microbiol
. 39: 71-81).
Transferrin-binding Proteins
Iron is essential for bacterial growth. However, it is complexed to the host glycoproteins transferrin and lactoferrin in the extracellular environment. Thus, the pathogenic bacteria have developed different strategies to acquire this element. Several Actinobacillus, Haemophilus and Pasteurella species have shown to possess a mechanism of iron acquisition involving direct binding of the hosts transferrin by receptor proteins on the surface. Those receptors are only expressed under iron restriction conditions. They consist of two distinct proteins, Tbp1 and Thp2, specific for host's transferrin. Tbp1 proteins are probably transmembrane proteins that serve as channels for transport of iron across the outer membrane, while Tbp2 seem to be lipoproteins anchored to the outer membrane by their N-terminal lipid tail. Schryvers (Schryvers, A. B. 1994. Abstracts Haemophilus, Actinobacillus and Pasteurella International Conference, Edinburgh, UK, 23-24) suggested an iron acquisition pathway involving the binding and removal of iron from transferrin at the bacterial surface by the coordinate action of Tbp1 and Thp2, followed by a transport of iron across the outer membrane via Tbp1 and binding of iron by a periplasmic binding protein.
Three other Thp proteins, of 60, 62 and 65 kDa, have been identified among App serotypes. Immunization of pigs with the 60-kDa Tbp conferred limited protection to the challenge with homologous strains (Gerlach, G. F., Anderson, C., Potter, A. A., Klashinsky, S. and Wilson, P. J. 1992
. Infect. Imm
. 60: 892-898).
Proteases
App secretes proteases that degrade porcine gelatin, immunoglobulin A (thereafter “IgA”) and haemoglobin (Negrete-Abascal, E., Tenorio, V.R., Serrano, J. J., Garcia, C., de la Garza, M. 1994
. A. Can. J. Vet Res
. 58: 83-86). It has been suggested that the cleavage of IgA by proteases could facilitate the mucosal spread of App, and that proteolysis of haemoglobin could be a mechanism of iron acquisition.
RTX toxins
Two haemolytic RTX exotoxins (I and II) and one cytolytic, non haemolytic RTX exotoxin (III) have been described among the different App serotypes (Frey, J., Bosse, J. T., Chang, Y., Cullen, J. M., Fenwick, B., Gerlach, G. F., Gygi, D., Haesebrouck, F., Inzana, T. J., Jansen, R., Kamp, E. M., Macdonald, J., Maclnnes, J. L., Mittal, K. R., Nicolet, J., Rycroft, A., Segers, R. P. A. M., Smits, M. A., Stenbaek, E., Struck, D. K., van den Bosch, J. F., Willson, P. J. and Young, R. 1993
. J. Gen. Microbiol
. 139: 1723-1728). They belong to the pore-forming RTX-toxin family (repeat-in-the-toxin), widely spread among pathogenic Gram-negative bacteria. These exotoxins are toxic for porcine alveolar macrophages and neutrophils.
The protection conferred by these toxins against porcine pleuropneumonia has been demonstrated by immunization of
Daban Montserrat
Espuña Enric
Medrano Andres
Querol Enrique
Graser Jennifer E.
Henderson & Sturm LLP
Laboratorios Hipra S.A.
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