Method for attenuating a bacterium of the mycobacterium tuberculosis complex for producing a tuberculosis vaccine

A technology of mycobacterium tuberculosis and mycobacteria, applied in the direction of microorganism-based methods, methods using microorganisms, biochemical equipment and methods, etc., can solve the problems of weakening growth and not effectively adding genes

Inactive Publication Date: 2015-01-07
FOND MEDITERRANEE INFECTION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these bioengineering techniques have not been effectively used to add genes missing from the bacterial genome of the Mycobacterium tuberculosis complex for the purpose of being used as a vaccine or producing a vaccine
[0010] 2- There are no prior art studies showing that the expression of the mspA gene can attenuate the growth of bacteria of the Mycobacterium tuberculosis complex in eukaryotic cell models and animal models, and why it can be used as a vaccine or to produce a vaccine

Method used

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  • Method for attenuating a bacterium of the mycobacterium tuberculosis complex for producing a tuberculosis vaccine
  • Method for attenuating a bacterium of the mycobacterium tuberculosis complex for producing a tuberculosis vaccine
  • Method for attenuating a bacterium of the mycobacterium tuberculosis complex for producing a tuberculosis vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1. Growth of Mycobacterium tuberculosis expressing MspA in sterile media

[0047] First, the inventors amplified the gene encoding the MspA porin from the Mycobacterium smegmatis genome, and then inserted the amplified product into the expression vector pVV16 [16] to obtain the plasmid pVVMspA. Two genes for proteins of kanamycin and kanamycin (selection markers). The inventors then incorporated the pVVMspA plasmid into the M. tuberculosis strain HS7Rv by electroporation, and screened transformed clones using hygromycin and kanamycin as antibiotics.

[0048] Experimental protocol

[0049] 1) Construction of pVVMspA plasmid:

[0050] First, the inventors extracted genomic DNA from M. smegmatis strain mc155 (ATCC 700084) and used standard PCR to amplify the gene encoding the MspA porin [5].

[0051] The sequence gi_118467340 of the gene is the following sequence SEQ.ID.N°1:

[0052] 5′-

[0053] ATGAAGGCAATCAGTCGGGTGCTGATCGCGATGGTTGCAGCCATCGCGGCGCTTTTCACGAGCA...

Embodiment 2

[0069] Example 2. Growth of co-cultures of Mycobacterium tuberculosis expressing MspA and the amoebae Acanthamoeba polyphaga

[0070] 1) Experimental procedures:

[0071] 10mL amoeba suspension (~10 5 Amoeba / mL) inoculation~10 6 Mycobacteria / ml (MOI=10). As a control, Acanthamoeba polyphagoides and mycobacteria were cultured separately in PAS buffer (Page's modified Neff's amoeba saline) (21, 22, 23). After an incubation period of 24 h at 32°C, the co-cultures were washed three times with PAS and incubated in 10 ml PAS for 10 to 15 minutes at 32°C.

[0072] 2) Results:

[0073] Using amoebae as eukaryotic cells, two strains of Mycobacterium tuberculosis H37Rv / pVV16 and Mycobacterium tuberculosis H37Rv / pVVMspA were observed to survive in the experiment after 8 days of co-culture, but the strains of Mycobacterium tuberculosis H37Rv / pVVMspA expressing the MspA porin The number of mycobacteria was significantly reduced (P≤0.05; Student's statistical test). On day 12, the num...

Embodiment 3

[0076] Example 3. Growth of co-cultures of M. tuberculosis expressing MspA and human macrophages (hMdM) or mouse macrophages (BMDM):

[0077] 1) Experimental procedures:

[0078] Using Ficoll gradients (MSL, Eurobio, Courtaboeuf France) from buffy coat (leukopack) (France, Marseille, National Blood Service (Etablissement du Sang)) to isolate BMDM[24] and hMdM, which were then differentiated, and respectively (10 5 cells / well) were seeded in 24-well culture plates in RPMI 1640 medium containing 10% FCS. BMDM and hMdM were infected with Mycobacterium tuberculosis, Mycobacterium tuberculosis H37Rv / pVV16, and Mycobacterium tuberculosis H37Rv / pVVMspA for 24 hours, respectively. Serum (FCS) was incubated in RPMI1640 medium for different periods of time. As a control, each cell type and mycobacteria were cultured separately.

[0079] Determine the number of intracellular mycobacteria (CFU):

[0080] At the given times, infected cells were lysed with 0.1% sodium dodecyl sulfat...

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Abstract

The present invention relates to the use of a strain of a mycobacterium of the Mycobacterium tuberculosis complex, into which an mspA gene capable of expressing a Mycobacterium smegmatis porin A has been inserted, for producing a vaccine for the prevention of an infection by a bacterium of the Mycobacterium tuberculosis complex in a host comprising eukaryotic cells, preferably macrophages, wherein said mycobacterial strain thus transformed exhibits reduced growth in said eukaryotic cells.

Description

Background technique [0001] Tuberculosis is an infectious disease affecting humans and animals due to the presence of one of the species, the Mycobacterium tuberculosis complexe, i.e. in particular Mycobacterium tuberculosis, Mycobacterium bovis ( Mycobacterium bovis) (and BCG strains derived therefrom), Mycobacterium africanum, Mycobacterium canettii, Mycobacterium caprae, Mycobacterium microti and Mycobacterium pinnipedii [19] and Mycobacterium mungii (20). Tuberculosis causes approximately 9 million new infections each year around the world and kills approximately 3 million people each year. The HIV infection epidemic has coincided with the re-emergence of tuberculosis in developed countries and, worryingly, has been accompanied by the emergence of Mycobacterium tuberculosis strains resistant to first-line anti-TB antibiotics. Tuberculosis is an infectious disease that can be transmitted from people with rod-shaped tuberculosis, ie, people who shed live mycobacteria of th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20A61K39/04C07K14/35C12R1/32
CPCC12N1/20A61K2039/522C07K14/35C12N1/36A61K39/04C12N1/205C12R2001/32A61P31/06A61P37/04
Inventor M·德朗古O·拉姆贝特D·拉乌尔
Owner FOND MEDITERRANEE INFECTION
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