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Live attenuated RTX-producing bacteria of family pasteurellaceae

A Pasteurella, RTX- technology, applied in bacteria, methods of using bacteria, virus antigen components, etc., can solve problems such as undescribed and lack of vaccination experiments

Inactive Publication Date: 2003-08-13
INTERVET INT BV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, vaccination experiments with toxin subunits have not been described
[0028] However, all RTX-toxin subunit vaccines suffer from three major drawbacks: A large amount of antigenic material is required to adequately stimulate the immune system
However live attenuated bacteria attenuated by loss of ability to express the RTX-toxin will substantially lack the most important virulence factor, the RTX-toxin, and thus will not elicit an immune response to the toxin

Method used

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  • Live attenuated RTX-producing bacteria of family pasteurellaceae
  • Live attenuated RTX-producing bacteria of family pasteurellaceae
  • Live attenuated RTX-producing bacteria of family pasteurellaceae

Examples

Experimental program
Comparison scheme
Effect test

Embodiment Embodiment 1

[0093] Example Example 1: Preparation of live attenuated strains of Actinobacillus pleuropneumoniae. Construction of pApxI-D11:

[0094] The feasibility of the live attenuated bacteria according to the invention was demonstrated by the construction of the ΔapxIC mutant of Actinobacillus pleuropneumoniae.

[0095] To obtain this mutant, a plasmid including a deletion in the RTX-activator gene was first prepared.

[0096] The build outline is shown in Figure 1. The sequence of the apxI operon of Actinobacillus pleuropneumoniae is deposited in GenBank with the accession number X52899. From pJFF750 (Gygi et al.; Infection Immunity 60:3059-3064, 1992), the apxIC and apxIA genes and promoter regions were excised into a 4481 bp BglII / PstI fragment and cloned into plasmid pGEM3Z (Promega Co., Leiden NL) to BamHI and PstI digestion. The resulting plasmid was named pApxI-D1. A DNA fragment of 407 bp including most of the ApxIC coding region was excised by digestion with restricti...

Embodiment 2

[0102] Most importantly, it has been shown that the SspI / XhoI fragment including the apxIC gene is absent in MBHPP113 ( figure 2 , right group). Example 2:ΔapxIC mutant MBHPP113 tested for loss of RTX-toxin expression, secretion and hemolytic activity. Loss of hemolytic activity in the Actinobacillus pleuropneumoniae ΔapxIC mutant MBHPP113.

[0103] To demonstrate the deletion effect of ΔapxIC in MBHPP113, the hemolytic activity of the ΔapxIC strain MBHPP113 and the wild-type parent strain were compared after cultured on blood plates.

[0104] A reference strain of A. pleuropneumoniae belonging to serotype 7 (producing ApxII only) was also included for comparison.

[0105] Since ApxII has moderate hemolytic activity, this strain exhibits a moderate level of hemolysis.

[0106] Using a sterile toothpick, transfer a single colony to a Columbia agar plate containing (0.1% NAD and 2% sheep red blood cells. result:

[0107] as from image 3 As can be seen in , after 8 hou...

Embodiment 3

[0115] The experimental results in Example 2 prove that the Actinobacillus pleuropneumoniae MBHPP113 strain can indeed express and export the inactive form of RTX-toxin. Example 3: Construction of ΔapxICΔapx IIC mutant from Actinobacillus pleuropneumoniae strain 4074

[0116] Construction of the ΔapxIC mutation in strain 4074 was done as described in Example 1. Isolation of Actinobacillus pleuropneumoniae following incubation of a serotype 1 reference strain (Frey and Nicolet, J. Clin. Microbiol. 28:232-236, 1990) in the presence of streptomycin and subsequently in the presence of both streptomycin and nalidixic acid Recipient strain MBHPP104. Following conjugation of MBHPP104 to MBHP101 (described in Example 1), A. pleuropneumoniae resistant to nalidixic acid and gentamicin was obtained. After plating this strain on CBM plates containing 2% sheep erythrocytes, 0.1% NAD and nalidixic acid, several colonies with smaller hemolytic bands were identified. One of these coloni...

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Abstract

The present invention relates to live attenuated RTX-toxin producing bacteria of the family Pasteurellaceae, of which the attenuation is due to the fact that they produce RTX toxin in a non-activated form. The invention also relates to vaccines for the protection of mammals against infection with RTX-toxin producing bacteria of the family Pasteurellaceae, and to methods for the preparation of said live attenuated bacteria and vaccines.

Description

technical field [0001] The present invention relates to live attenuated RTX-producing Pasteurellaceae bacteria, methods of producing such bacteria, vaccines comprising such bacteria, methods of producing such vaccines and protection of humans and animals from pathogenic A method of infection with RTX-producing Pasteurellaceae bacteria. The Pasteurella family includes the genera Haemophilus, Actinobacillus and Pasteurella. This family of bacteria is also frequently referred to as HAP-group bacteria. Several species of these closely related genera are known to express a homologous calcium-dependent pore-forming cytotoxin, the so-called RTX toxin. (RTX stands for repeat in toxin). RTX-toxin-producing bacteria of this family are responsible for a whole field of infectious diseases, affecting humans and animals. Background technique [0002] Other genera not related to the HAP-group, such as Escherichia and Bordetella, are also known to have RTX-toxins. These RTX-toxins are ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20A61K39/00A61K39/102A61K39/12A61K39/135A61K39/145A61K39/15A61K39/155A61K39/295A61K39/39C07K14/285
CPCC12R1/21C12R1/01C07K14/285A61K39/295A61K39/00C12R2001/21C12N1/205C12R2001/01C12N1/20C12P1/04A61K39/102C12N15/01
Inventor R·P·A·M·塞格斯J·F·博希·宛·登J·弗雷
Owner INTERVET INT BV
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