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Tuberculosis vaccines including recombinant bcg strains overexpressing phop, and/or phop regulon protein(s)

A technology of overexpression and bacterial strains, applied in the field of recombinant BCG strains, can solve the problems of loss of immune protection and weakened potency

Active Publication Date: 2012-05-02
成都安永鼎业生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Existing BCG vaccines confer protection against manifestations of tuberculosis in children, but their efficacy wanes over a period of 10 to 15 years, presumably because of the gradual loss of BCG-induced immune protection [13]

Method used

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  • Tuberculosis vaccines including recombinant bcg strains overexpressing phop, and/or phop regulon protein(s)
  • Tuberculosis vaccines including recombinant bcg strains overexpressing phop, and/or phop regulon protein(s)
  • Tuberculosis vaccines including recombinant bcg strains overexpressing phop, and/or phop regulon protein(s)

Examples

Experimental program
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preparation example Construction

[0063] The preparation of recombinant live vaccines is known to those skilled in the art. Typically the vaccines are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared. The formulation can also be emulsified, or the protein encapsulated in liposomes. The live immunogenic ingredient is usually mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, etc., and combinations thereof. In addition, if desired, the vaccine may contain minor amounts of auxiliary substances, such as wetting or emulsifying agents, pH buffering agents and / or adjuvants which enhance the efficacy of the vaccine. Examples of potentially effective adjuvants include, but are not limited to: aluminum hydroxide, N-acetylmuramoyl-L-threonyl-D-isoglutamine (thr-MDP), ...

Embodiment 1

[0074] Example 1. Construction of BCG strains overexpressing the transcription regulator protein phoP

[0075] A kanamycin-resistant shuttle vector containing the T7 promoter was obtained by cutting the pDrive cloning vector (obtained from Qiagen) with EcoRI and generating pDRI by self-ligation. pDRI was digested with SspI and the 1903 base pair fragment product was isolated. The pMD31 shuttle vector (Wu et al., 1993, Molecular Microbiology, 7, 407-417) was digested with SspI and a fragment of 3379 base pairs was isolated. The ligation of these two SspI-generated fragments produces pME (5282 bp), which contains the original T7 promoter of pDRIVE (see image 3 ).

[0076] Forward primers (5'-AAAAA) containing KpnI and pstI restriction sites (underlined parts) respectively GGTACC GCTTGTTTGGCCATGTCAAC-3') and reverse primer (5'-AAAAA CTGCAG GCTGCCGATCCGATTAACTAC-3'), the wild-type M. tuberculosis phoP gene was amplified from the M. tuberculosis H37Rv strain (ATCC 25618). T...

Embodiment 2

[0078] Example 2. Determination of Induction of Recombinant BCG-Prague Genes Overexpressing phoP

[0079] To determine the effect of phoP overexpression on BCG gene expression, microarray analysis was performed to compare the transcriptional profiles of BCG-Prague / pME:phoP and wild-type BCG-Prague harboring pME empty vector. The strain was cultured at 37°C in 7H9 liquid medium supplemented with 10% ADC (Difco) and 0.05% Tween 80 until OD 600 Values ​​reach 0.4 to 0.5. To isolate total RNA, cells were pelleted and transferred to 2 ml screw cap tubes containing 1 ml RNA protect Bacterial Reagent (Qiagen) and incubated for 5 min at room temperature. The cells were pelleted again, and 400 μl of lysate (20 mM NaCH 3 COOH, 0.5% SDS, 1 mM EDTA, pH 4) and 1 ml phenol / chloroform (pH 4.5, Sigma) were resuspended. Cells were disrupted by beating with glass beads in three 30 sec-pulses. Cells were then incubated at 65°C for 4 minutes, followed by 4°C for 5 minutes and centrifuged at 1...

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Abstract

A live recombinant Mycobacterium bovis-BCG strain comprising a nucleic acid encoding PhoP protein and / or one or more phoP regulon proteins is provided, in which the nucleic acid is capable of being overexpressed. A vaccine or immunogenic composition comprising the live recombinant Mycobacterium bovis-BCG strain and a method for treatment or prophylaxis of a mammal against challenge by Mycobacterium tuberculosis or Mycobacterium bovis are also provided.

Description

field of invention [0001] The present invention relates to tuberculosis (TB) vaccines. Specifically, the present invention provides recombinant BCG (BCG) strains that overexpress the transcription factor PhoP at a level sufficient to cause the phoP regulon to be induced. Background of the invention [0002] Tuberculosis, caused by Mycobacterium tuberculosis (M.tb), has been a global health emergency. According to the latest surveillance data from the World Health Organization (WHO), there were 9.2 million new cases and 1.7 million deaths from tuberculosis in 2006. The challenges of TB prevention, case detection, and treatment are in stark contrast to the ease with which the bacilli spread. Other threats to TB control include the spread of multidrug-resistant TB (MDR-TB), the emergence of extensively drug-resistant TB (XDR-TB), and the devastating impact of TB / HIV co-infection. Due to these circumstances, there is an urgent need for effective alternatives to antibiotics to...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/31C07K14/35C12R1/32A61K39/04A61P31/06A61P35/00
CPCC07K14/35A61K2039/52A61K39/04A61P31/06A61P35/00
Inventor 刘军
Owner 成都安永鼎业生物技术有限公司
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