Development of a New Approach to Determine the Potency of Bacille Calmette–Guérin Vaccines Using Flow Cytometry

Gweon, Eunjeong; Choi, Chan-Woong; Kim, Jaeok; Kim, Byung-Kuk; Kang, Hyun Kyung; Park, Taejun; Ban, Sangja; Bae, Minseok; Park, Sangjin; Jeong, Jayoung

doi:10.24171/j.phrp.2017.8.6.06

Cited by 6 publications

(9 citation statements)

References 9 publications

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“…However, this method is highly subjective. In 2017, Gweon et al demonstrated that the CV% between different laboratories can be as high as 95% (with an average of 49%) when using CFU assay for analysis 20 .…”

Section: Discussionmentioning

confidence: 99%

“…Recently, flow cytometry assays were developed based on the use of highly sensitive laser-based flow cytometer technology for cell counting 14 , 20 . In this case, two strategies can be employed, known as dye-exclusion and dye-uptake 21 .…”

Section: Introductionmentioning

confidence: 99%

“…Yang et al 14 employed the dye-exclusion strategy using PI for determining the percentage of dead cells in the BCG vaccine 14 . On the other hand, Gweon et al 20 used the dye-uptake strategy for direct staining of living cells with FD for measuring the percentage of living cells 20 . In both cases, the flow cytometer was coupled to a Coulter counter for counting the total number of cells (living and dead); multiplying this number by the percentage of living/dead cells yields the absolute number of the corresponding cells 14 , 20 .…”

Section: Introductionmentioning

confidence: 99%

“…On the other hand, Gweon et al 20 used the dye-uptake strategy for direct staining of living cells with FD for measuring the percentage of living cells 20 . In both cases, the flow cytometer was coupled to a Coulter counter for counting the total number of cells (living and dead); multiplying this number by the percentage of living/dead cells yields the absolute number of the corresponding cells 14 , 20 . However, these studies require the use of multiple apparatus making it liable to handling variations.…”

Section: Introductionmentioning

confidence: 99%

“…However, these studies require the use of multiple apparatus making it liable to handling variations. Moreover, the correlation of these assays with CFU assay was variable and ranged from 0.7 to 0.95 14 , 20 .…”

Section: Introductionmentioning

confidence: 99%

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Development of a fast and precise potency test for BCG vaccine viability using flow cytometry compared to MTT and colony-forming unit assays

Moghawry,

Rashed,

Gomaa

et al. 2023

Sci Rep

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In a precarious world of rapidly growing pandemics, the field of vaccine production has witnessed considerable growth. Bacillus Calmette-Guérin (BCG) is a live-attenuated vaccine and a part of the immunization program in 157 countries. The quality control is based on a potency test through viable cell enumeration. The colony-forming unit (CFU) assay is the official method, however, it often yields fluctuating results, suffers from medium cracking, and requires lengthy analysis (~ 28 days). Flow cytometric analysis was proposed earlier, but it was coupled with a Coulter counter for measuring the entire bacterial population (live/dead). In the present study, thiazole orange/propidium iodide dyes supplemented with fluorogenic reference beads were employed for viable counting, eliminating the need for a Coulter counter. Both the flow cytometry and the colorimetric technique employing tetrazolium salt were validated and compared to the CFU assay. The colorimetric assay displayed high precision, accuracy, and a strong positive correlation with the CFU assay. The flow cytometry assay demonstrated high precision and a notable ability to distinguish different forms of BCG cells (live, injured, and dead). It also exhibited a perfect positive correlation with the CFU assay. Both methods reduced the analysis time by > 26 days and eliminated the need for human intervention by automating the test.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Development of a fast and precise potency test for BCG vaccine viability using flow cytometry compared to MTT and colony-forming unit assays

Moghawry,

Rashed,

Gomaa

et al. 2023

Sci Rep

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Recent Developments in the Application of Flow Cytometry to Advance our Understanding of Mycobacteriumtuberculosis Physiology and Pathogenesis

2020

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The ability of the bacterial pathogen Mycobacterium tuberculosis to adapt and survive within human cells to disseminate to other individuals and cause active disease is poorly understood. Research supports that as M. tuberculosis adapts to stressors encountered in the host, it exhibits variable physiological and metabolic states that are time and niche‐dependent. Challenges associated with effective treatment and eradication of tuberculosis (TB) are in part attributed to our lack of understanding of these different mycobacterial phenotypes. This is mainly due to a lack of suitable tools to effectively identify/detect heterogeneous bacterial populations, which may include small, difficult‐to‐culture subpopulations. Importantly, flow cytometry allows rapid and affordable multiparametric measurements of physical and chemical characteristics of single cells, without the need to preculture cells. Here, we summarize current knowledge of flow cytometry applications that have advanced our understanding of the physiology of M. tuberculosis during TB disease. Specifically, we review how host‐associated stressors influence bacterial characteristics such as metabolic activity, membrane potential, redox status and the mycobacterial cell wall. Further, we highlight that flow cytometry offers unprecedented opportunities for insight into bacterial population heterogeneity, which is increasingly appreciated as an important determinant of disease outcome. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

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The Role of Fermentation in BCG Manufacture: Challenges and Ways Forward

Walker¹,

Bacon²

2023

Vaccines for Neglected Pathogens: Strategies, Achievements and Challenges

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The BCG vaccine has been in existence for a century and has been instrumental in the control of tuberculosis. The method for producing the vaccine has not changed in a very long time and consists of pellicle growth followed by ball-milling, which is a lengthy and variable approach. There has been increasing interest in the possibility of producing the BCG vaccine by growing it in bioreactors, which could address some of the issues around variation between batches, increase yield, and circumvent the challenges associated with supply and demand. There is evidence that fermentation would be a quicker, more reproducible method of production, and would deliver BCG to a higher yield in a form that would be easier to characterise. However, a change to the manufacturing process may require new evidence of bioequivalence and may attract a requirement for preclinical studies as well as clinical trials from Phase I, through to efficacy studies. This chapter describes the history of the BCG vaccine and the issues of the current production method. We discuss the potential benefits of BCG fermentation and the regulatory steps required for such a method of production to be implemented.

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Development of a New Approach to Determine the Potency of Bacille Calmette–Guérin Vaccines Using Flow Cytometry

Cited by 6 publications

References 9 publications

Development of a fast and precise potency test for BCG vaccine viability using flow cytometry compared to MTT and colony-forming unit assays

Development of a fast and precise potency test for BCG vaccine viability using flow cytometry compared to MTT and colony-forming unit assays

Recent Developments in the Application of Flow Cytometry to Advance our Understanding of Mycobacteriumtuberculosis Physiology and Pathogenesis

The Role of Fermentation in BCG Manufacture: Challenges and Ways Forward

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