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

David Pires; Paulo J. G. Bettencourt

doi:10.3791/65447

Journal

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Medicine

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

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Medicine

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JoVE Journal Medicine

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

2,046 Views

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06:26 min

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July 28, 2023

DOI:

10.3791/65447-v

DOI:

10.3791/65447-v

•

06:26 min

July 28, 2023

•

19 Views

David Pires^1,2,3, Paulo J. G. Bettencourt^1,2

¹Faculty of Medicine,Universidade Católica Portuguesa, ²Center for Interdisciplinary Research in Health,Universidade Católica Portuguesa, ³Host-Pathogen Interactions Unit, Research Institute for Medicines, iMed-ULisboa, Faculty of Pharmacy,Universidade de Lisboa

Transcript

CFU determination is necessary to measure the efficacy of the immune response against bacteria. However, it is very low throughput and becomes cumbersome and expensive when applied to pathogenic bacteria such as Mycobacterium tuberculosis. This is a significant limitation when we need to test large panels of treatments.

The research focuses on tuberculosis and on the assessments of TB vaccines. It explores safety, immunogenicity, and efficacy of vaccines against Mycobacterium tuberculosis. The study presents an optimized method to micro-CFU for enumerating bacterial colonies to assess vaccine efficacy in preclinical studies.

This protocol aims to optimize CFU determination by generating results from a small droplet similar to those from a regular Petri dish. Quantifying bacteria viability from such a small sample allows us to analyze thousands of data points using a fraction of the materials and time necessary for the classical method. Our research aims to dissect the antigen presentation machinery and to identify the most relevant antigens to optimize antigen presentation.

This will inform us to design and develop better vaccines to improve or replace the current TB vaccine, which is BCG.

Summary

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.