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Mycobacterium mutants for vaccines with improved protective efficacy

一种分枝杆菌、牛分枝杆菌的技术，应用在疫苗领域

Inactive Publication Date: 2015-03-11
VLAAMS INTERUNIVERSITAIR INST VOOR BIOTECHNOLOGIE VZW +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Vaccines with attenuated M. tuberculosis also have potential safety concerns and special care must be taken (eg, double mutations, each single mutation resulting in sufficient attenuation) (Kamath et al., 2005; Walker et al., 2010)

Method used

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  • Mycobacterium mutants for vaccines with improved protective efficacy
  • Mycobacterium mutants for vaccines with improved protective efficacy
  • Mycobacterium mutants for vaccines with improved protective efficacy

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0153] Example 1. Generation and screening of M. bovis BCG mutants

[0154] Transposon delivery by fungal phage is an efficient method for generating mycobacterial saturation mutagenesis libraries (54). This method makes it possible to generate multiple mutants in an isogenic background suitable for comparing the effects of different genetic mutations. Insertion of a transposon into a gene most often results in its inactivation, and the frequency of reversion is usually very low (47). Transposons can also be used to deliver other markers, such as antibiotic resistance genes. It also allows transposons to flank genomic regions to be amplified and identified by sequencing (27). Transposon mutagenesis has been used successfully in both fast-growing and slow-growing mycobacterial strains (1, 10, 32, 51). Here, we generated a 96 x 96 clone ordered library of M. bovis BCG transposon insertion mutants using Himarl-derived minitransposons (49) delivered by temperature-sensitive Myc...

Embodiment 2

[0160] Example 2. Biochemical characterization of transposon insertion mutants

[0161] Southern analysis to confirm single transposon insertion

[0162]The presence of a single transposon insertion in the genome was confirmed by further characterization of sequence-confirmed mutants by Southern blotting probed with the full-length transposon. This is essential to ensure that the mutant phenotypes observed are caused by transposon insertions that result in the inactivation of the gene of interest alone. Except for the three mutants ( figure 2 Except for A), all the remaining mutants showed single transposon insertion.

[0163] Mutants in Mb1661c, Mb2203, Mb2196 and SapM

[0164] In our library screening, we obtained 2 mutants each for the genes Mb1661c, Mb2203 and Mb2196. One of the mutants of the genes Mb2203 and Mb2196 showed multiple transposon insertions when analyzed by Southern blot analysis and was removed from further use. Of the two mutants of the gene Mb1661c, ...

Embodiment 3

[0184] Example 3. Protective efficacy of Mycobacterium bovis BCG wild type against mutants SapM::T(Mb3338::T), Mb2203::T, Mb1661c::T and Mb2196::T in BALB / c mice

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Abstract

Tuberculosis (TB) is a major health problem and currently, the only licensed TB vaccine is Mycobacterium bovis Bacille Calmette-Guerin (M. bovis BCG). In the present invention, mutation of mycobacterial components reportedly involved in phagosome maturation inhibition was evaluated for vaccine purposes, as such mutations should result in better vaccine antigen processing and presentation. Thus, BCG mutants in genes coding for ManLAM capping α-1,2-mannosyltransferases and the PI3P phosphatase SapM were evaluated as TB vaccines in a stringent mouse model. Vaccination with both ManLAM capping mutants and the SapM mutant resulted in significantly longer survival as compared to non-vaccinated mice, whereas vaccination with the parental BCG did not. Moreover, mice vaccinated with the SapM mutant survived significantly longer than mice vaccinated with the parental BCG. The mutant BCG strains showed unaltered phagocytosis, replication, lysosome colocalization and oxidant activity in macrophages and similarly induced autophagy in the latter. Additionally, replication and granuloma formation in mice was unaffected, indicating BCG-equivalent safety of these vaccines.

Description

technical field [0001] The present invention relates to the field of vaccines, most particularly vaccines based on live attenuated Mycobacterium. Disclosed herein are genes that, when mutated in mycobacteria used to immunize animals, especially mammals, confer improved protective efficacy as a vaccine against Mycobacterium infection, while not interfering with branches such as bovine Beneficial properties of known inoculated strains of Mycobacterium bovis BCG. Such vaccines are particularly suitable for Mycobacterium tuberculosis infection. Background technique [0002] One third of the world's population is infected with the TB pathogen Mycobacterium tuberculosis (M.tb). The World Health Organization estimates that between eight and ten million new TB cases occur worldwide each year, and the incidence of TB is currently increasing. Along with malaria and HIV-AIDS, TB is one of the top three causes of death from a single infectious agent, with approximately two million de...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K39/04C12N9/10C12N9/16A61K35/74A61K39/00
CPCA61K2039/522C12N9/16A61K39/04C12N9/1051A61P31/04A61P31/06
Inventor N·卡勒维尔特A·巴特尼N·菲斯詹斯C·许根
Owner VLAAMS INTERUNIVERSITAIR INST VOOR BIOTECHNOLOGIE VZW
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