bacillus calmette-gu&#233;rin (bcg) vaccine production

使用 Micro-CFU 方法加速结核病测定

Tuberculosis (TB) is the leading cause of death worldwide by a single infectious agent, bacterium Mycobacterium tuberculosis (M.tb), killing more people than any other pathogen. In 2021, TB was responsible for 1.6 million deaths and was surpassed by COVID-19 during the 2019-2021 pandemic1. Moreover, according to the World Health Organization&#180;s global TB report of 2022, the COVID-19 pandemic was responsible for an increase in new TB cases. The WHO also reports large drops in the number of people diagnosed with TB during this period, which could increase further the number of TB cases1.
The Bacillus Calmette-Gu&#233;rin (BCG) is a live-attenuated strain of the pathogenic Mycobacterium bovis, used for the first time as a vaccine more than 100 years ago. This is the only vaccine against TB and is the oldest licensed vaccine in the world still in use2,3. Currently, there are 12 vaccines in different phases of clinical trials4, and dozens of vaccines are under pre-clinical development5,6. Pre-clinical assessment of vaccines against TB includes the evaluation of the safety and immunogenicity7, which can be obtained in diverse animal models such as zebrafish, mice, guinea pigs, rabbits, cattle, and non-human primates8,9,10. Additionally, assessing the capacity of a vaccine to induce protection against M.tb infection and/or transmission, i.e., the vaccine efficacy, requires an M.tb challenge in vivo5,11. Interestingly, BCG vaccination induces non-specific effects that affect the survival of other bacterial and viral pathogens12,13&#160;through the mechanism of trained immunity14. To quantify the viable bacterial burden in an infected animal, the method of choice is the enumeration of bacterial colonies through the colony-forming units (CFU) assay5,15. CFU is a unit that estimates the number of microorganisms (bacteria or fungi) that form colonies under specific growth conditions. CFUs originate from viable and replicative microorganisms, and the absolute number of living microorganisms within each colony is difficult to estimate. It is uncertain whether a colony has originated from one or more microorganisms. The CFU unit reflects this uncertainty, hence a great variability can be observed in replicates of the same sample. This time-consuming assay requires specialized technicians trained to work in a biosafety level 3 (BSL3) facility, substantial laboratory and incubator space, takes from 4 to 6 weeks to conclude, and is prone to contamination.
In this study, we describe an optimized method for colony enumeration, the micro-CFU (mCFU), and offer a simple and rapid solution to analyze the results15,16,17,18,19,20. The mCFU assay requires tenfold fewer reagents, reduces the incubation period threefold, taking 1 to 2 weeks to conclude, reduces lab space and reagent cost, and minimizes the health and safety risks associated with working with large numbers of M.tb. We propose that this method should be universally adopted for pre-clinical studies of vaccine efficacy determination, ultimately leading to time reduction in the development of vaccines against TB. Finally, this optimized method of CFU enumeration has been used to quantify not only Mycobacteria but also other bacteria, such as Escherichia coli and Ralstonia solanacearum21.

作者聚焦：优化 CFU 测定以有效评估结核病疫苗功效和抗原呈报分析 

用于结核病疫苗功效评估的微集落形成单位测定

Watch this Scientific Journal Video about Bacillus Calmette-GuÃ©rin BCG Vaccine Production at JoVE.com

Bacillus Calmette-Gu&#233;rin (BCG) Vaccine Production  

卡介苗（BCG）疫苗生产

首先在对数生长的中期或晚期收集 BCG 培养物。离心3000G10分钟，然后弃去上清液。接下来，加入 10 毫升 PBS 清洗细菌。离心后，弃去上清液。将沉淀重悬于五毫升感染培养基中。将管子置于超声浴中以全功率15分钟，频率为80赫兹。现在以 1， 000 g 离心 10 分钟。收集上清液并丢弃沉淀。测量上清液的光密度。

bacillus-calmette-gurin-bcg-vaccine-production

Research

JoVE Journal

Medicine

Bacillus Calmette-Gu&#233;rin (BCG) Vaccine Production

261 次观看. 首先在对数生长的中期或晚期收集 BCG 培养物。离心3000G10分钟，然后弃去上清液。接下来，加入 10 毫升 PBS 清洗细菌。离心后，弃去上清液。将沉淀重悬于五毫升感染培养基中。将管子置于超声浴中以全功率15分钟，频率为80赫兹。现在以 1， 000 g 离心 10 分钟。收集上清液并丢弃沉淀。测量上清液的光密度。

视频: Bacillus Calmette-Guérin (BCG) Vaccine Production  

Micro-Colony Forming Unit Assay for Efficacy Evaluation of Vaccines Against Tuberculosis

Tuberculosis (TB), the leading cause of death worldwide by an infectious agent, killed 1.6 million people in 2022, only being surpassed by COVID-19 during the 2019-2021 pandemic. The disease is caused by the bacterium Mycobacterium tuberculosis (M.tb). The Mycobacterium bovis strain Bacillus Calmette-Gu&#233;rin (BCG), the only TB vaccine, is the oldest licensed vaccine in the world, still in use. Currently, there are 12 vaccines in clinical trials and dozens of vaccines under pre-clinical development. The method of choice used to assess the efficacy of TB vaccines in pre-clinical studies is the enumeration of bacterial colonies by the colony-forming units (CFU) assay. This time-consuming assay takes 4 to 6 weeks to conclude, requires substantial laboratory and incubator space, has high reagent costs, and is prone to contamination. Here we describe an optimized method for colony enumeration, the micro-CFU (mCFU), that offers a simple and rapid solution to analyze M.tb vaccine efficacy results. The mCFU assay requires tenfold fewer reagents, reduces the incubation period threefold, taking 1 to 2 weeks to conclude, reduces lab space and reagent cost, and minimizes the health and safety risks associated with working with large numbers of M.tb. Moreover, to evaluate the efficacy of a TB vaccine, samples may be obtained from a variety of sources, including tissues from vaccinated animals infected with Mycobacteria. We also describe an optimized method to produce a unicellular, uniform, and high-quality mycobacterial culture for infection studies. Finally, we propose that these methods should be universally adopted for pre-clinical studies of vaccine efficacy determination, ultimately leading to time reduction in the development of vaccines against TB.

The determination of colony-forming units (CFU) is the gold-standard technique for quantifying bacteria, including Mycobacterium tuberculosis which can take weeks to form visible colonies. Here we describe a micro-CFU for CFU determination with increased time efficiency, reduced lab space and reagent cost, and scalability to medium and high throughput experiments.

Tuberculosis (TB), the leading cause of death worldwide by an infectious agent, killed 1.6 million people in 2022, only being surpassed by COVID-19 during ...