Name: Optimizing CFU Determination for Efficient Assessment of TB Vaccine Efficacy and Antigen Presentation Analysis (Video) | JoVE
Uploaded: 2023-07-28T13:10:00
Description: Watch this Scientific Journal Video about Optimizing CFU Determination for Efficient Assessment of TB Vaccine Efficacy and Antigen Presentation Analysis at JoVE.com

This work was supported by internal funding from the Faculty of Medicine, Universidade Cato&#769;lica Portuguesa, and external funding from Funda&#231;&#227;o para a Ci&#234;ncia e a Tecnologia (FCT), under the grants UIDP/04279/2020, UIDB/04279/2020, and EXPL/SAU-INF/0742/2021.

NOTE: The protocol described here is for BCG but can be applied to any Mycobacteria. BCG can be used as a surrogate bacterium for TB experiments when BSL3 facilities are not available22. The following procedures using BCG should be performed under a biosafety level 2 (BSL2) laboratory and follow the appropriate biosafety guidelines and good laboratory practices for the manipulation of hazard group 2 microorganisms.
1. Culture media preparation
<ol>
	<...

Accelerating Tuberculosis Determination Using the Micro-CFU Method

<ol>
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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+100th+anniversary+of+bacille+Calmette-Gu&#233;rin+(BCG)+and+the+latest+vaccines+against+COVID-19.">The 100th anniversary of bacille Calmette-Gu&#233;rin (BCG) and the latest vaccines against COVID-19.</a> The International Journal of Tuberculosis and Lung Disease. 25 (8), 611-613 (2021).</li><li>Scriba, T. J., Netea, M. G., Ginsberg, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Key+recent+advances+in+TB+vaccine+development+and+understanding+of+protective+immune+responses+against+Mycobacterium+tuberculosis.">Key recent advances in TB vaccine development and understanding of protective immune responses against Mycobacterium tuberculosis.</a> Seminars in Immunology. 50, 101431 (2020).</li><li>McShane, H., Williams, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+preclinical+animal+models+utilised+for+TB+vaccine+evaluation+in+the+context+of+recent+human+efficacy+data.">A review of preclinical animal models utilised for TB vaccine evaluation in the context of recent human efficacy data.</a> Tuberculosis. 94 (2), 105-110 (2014).</li><li>Voss, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+and+challenges+in+TB+vaccine+development.">Progress and challenges in TB vaccine development.</a> F1000Research. 7, 199 (2018).</li><li>Satti, I., McShane, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+approaches+toward+identifying+a+correlate+of+immune+protection+from+tuberculosis.">Current approaches toward identifying a correlate of immune protection from tuberculosis.</a> Expert Review of Vaccines. 18 (1), 43-59 (2019).</li><li>Young, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+tuberculosis.">Animal models of tuberculosis.</a> European Journal of Immunology. 39 (8), 2011-2014 (2009).</li><li>Pedroza-Rold&#225;n, C., Flores-Valdez, M. 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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevention+of+tuberculosis+in+macaques+after+intravenous+BCG+immunization.">Prevention of tuberculosis in macaques after intravenous BCG immunization.</a> Nature. 577 (7788), 95-102 (2020).</li><li>Madura Larsen, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BCG+stimulated+dendritic+cells+induce+an+interleukin-10+producing+T-cell+population+with+no+T++helper+1+or+T+helper+2+bias+in+vitro.">BCG stimulated dendritic cells induce an interleukin-10 producing T-cell population with no T helper 1 or T helper 2 bias in vitro.</a> Immunology. 121 (2), 276-282 (2007).</li><li>Bickett, T. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterizing+the+BCG-Induced+Macrophage+and+Neutrophil+Mechanisms+for+Defense+Against+Mycobacterium+tuberculosis.">Characterizing the BCG-Induced Macrophage and Neutrophil Mechanisms for Defense Against Mycobacterium tuberculosis.</a> Frontiers in immunology. 11, 1202 (2020).</li><li>Pires, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interference+of+Mycobacterium+tuberculosis+with+the+endocytic+pathways+on+macrophages+and+dendritic+cells+from+healthy+donors:+role+of+cathepsins.">Interference of Mycobacterium tuberculosis with the endocytic pathways on macrophages and dendritic cells from healthy donors: role of cathepsins.</a> Drug Discovery Today. 15 (23-24), 1112-1112 (2010).</li><li>Betts, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimising+Immunogenicity+with+Viral+Vectors:+Mixing+MVA+and+HAdV-5+Expressing+the+Mycobacterial+Antigen+Ag85A+in+a+Single+Injection.">Optimising Immunogenicity with Viral Vectors: Mixing MVA and HAdV-5 Expressing the Mycobacterial Antigen Ag85A in a Single Injection.</a> PLoS ONE. 7 (12), e50447 (2012).</li><li>Tanner, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+haemoglobin+and+iron+on+in+vitro+mycobacterial+growth+inhibition+assays.">The influence of haemoglobin and iron on in vitro mycobacterial growth inhibition assays.</a> Scientific reports. 7 (1), 43478 (2017).</li><li>McNeill, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+mycobacterial+infection+by+macrophage+Gch1+and+tetrahydrobiopterin.">Regulation of mycobacterial infection by macrophage Gch1 and tetrahydrobiopterin.</a> Nature communications. 9 (1), 5409 (2018).</li><li>Schindelin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nature Methods. 9 (7), 676-682 (2012).</li><li>Pereira, M., Vale, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Saquinavir:+From+HIV+to+COVID-19+and+Cancer+Treatment.">Saquinavir: From HIV to COVID-19 and Cancer Treatment.</a> Biomolecules. 12 (7), 944 (2022).</li><li>Pires, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repurposing+Saquinavir+for+Host-Directed+Therapy+to+Control+Mycobacterium+Tuberculosis+Infection.">Repurposing Saquinavir for Host-Directed Therapy to Control Mycobacterium Tuberculosis Infection.</a> Frontiers in immunology. 12, 647728 (2021).</li><li>Pires, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liposomal+Delivery+of+Saquinavir+to+Macrophages+Overcomes+Cathepsin+Blockade+by+Mycobacterium+tuberculosis+and+Helps+Control+the+Phagosomal+Replicative+Niches.">Liposomal Delivery of Saquinavir to Macrophages Overcomes Cathepsin Blockade by Mycobacterium tuberculosis and Helps Control the Phagosomal Replicative Niches.</a> International journal of molecular sciences. 24 (2), (2023).</li><li>Maartens, G., Wilkinson, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tuberculosis.">Tuberculosis.</a> The Lancet. 370 (9604), 2030-2043 (2007).</li><li>Matarazzo, L., Bettencourt, P. J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mRNA+vaccines:+a+new+opportunity+for+malaria,+tuberculosis+and+HIV.">mRNA vaccines: a new opportunity for malaria, tuberculosis and HIV.</a> Frontiers in Immunology. 14, 1172691 (2023).</li><li>Young, D., Dye, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Development+and+Impact+of+Tuberculosis+Vaccines.">The Development and Impact of Tuberculosis Vaccines.</a> Cell. 124 (4), 683-687 (2006).</li><li>Kommareddi, S., Abramowsky, C. R., Swinehart, G. L., Hrabak, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nontuberculous+mycobacterial+infections:+Comparison+of+the+fluorescent+auramine-o+and+Ziehl-Neelsen+techniques+in+tissue+diagnosis.">Nontuberculous mycobacterial infections: Comparison of the fluorescent auramine-o and Ziehl-Neelsen techniques in tissue diagnosis.</a> Human Pathology. 15 (11), 1085-1089 (1984).</li><li>Sabiiti, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Tuberculosis+Molecular+Bacterial+Load+Assay+(TB-MBLA).">A Tuberculosis Molecular Bacterial Load Assay (TB-MBLA).</a> Journal of visualized experiments: JoVE. (158), e60460 (2020).</li><li>Somosko&#776;vi, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+Recoveries+of+Mycobacterium+tuberculosis+Using+the+Automated+BACTEC+MGIT+960+System,+the+BACTEC+460+TB+System,+and+Lo&#776;wenstein-Jensen+Medium.">Comparison of Recoveries of Mycobacterium tuberculosis Using the Automated BACTEC MGIT 960 System, the BACTEC 460 TB System, and Lo&#776;wenstein-Jensen Medium.</a> Journal of Clinical Microbiology. 38 (6), 2395-2397 (2000).</li><li>Tanner, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+in+vitro+direct+mycobacterial+growth+inhibition+assay+(MGIA)+for+the+early+evaluation+of+TB+vaccine+candidates+and+assessment+of+protective+immunity:+a+protocol+for+non-human+primate+cells.">The in vitro direct mycobacterial growth inhibition assay (MGIA) for the early evaluation of TB vaccine candidates and assessment of protective immunity: a protocol for non-human primate cells.</a> F1000Research. 10, 257 (2021).</li></ol>

TB is an important public health problem with increasing importance, particularly in low and middle-income countries. The disruption of healthcare settings to diagnose and treat TB during the COVID-19 pandemic caused a negative impact on the incidence of new cases1. In addition, the multi-drug and extensively-drug resistant M.tb strains, and the co-infection of M.tb and HIV must be urgently addressed to control this epidemic1,3...

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>96-well plates</td>
			<td>VWR</td>
			<td>734-2781</td>
			<td></td>
		</tr>
		<tr>
			<td>DSLR 15-55 mm lens</td>
			<td>Nikon</td>
			<td>AF-P DX NIKKOR 18-55mm f/3.5-5.6G VR</td>
			<td></td>
		</tr>
		<tr>
			<td>DSLR camera</td>
			<td>Nikon</td>
			<td>D3400</td>
			<td></td>
		</tr>
		<tr>
			<td>DSLR macro lens</td>
			<td>Sigma</td>
			<td>MACRO 105mm F2.8 EX DG OS HSM</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal calf serum</td>
			<td>Gibco</td>
			<td>10270106</td>
			<td></td>
		</tr>
		<tr>
			<td>Fiji Software</td>
			<td></td>
			<td>https://fiji.sc/</td>
			<td>Fiji is an open-source software supported by several laboratories, institutions, and individuals. All the required plugins are included.</td>
		</tr>
		<tr>
			<td>Igepal CA-630</td>
			<td>Sigma-Aldrich</td>
			<td>18896</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Gibco</td>
			<td>25030-081</td>
			<td></td>
		</tr>
		<tr>
			<td>Middlebrook 7H10</td>
			<td>BD</td>
			<td>262710</td>
			<td></td>
		</tr>
		<tr>
			<td>Middlebrook 7H9</td>
			<td>BD</td>
			<td>271310</td>
			<td></td>
		</tr>
		<tr>
			<td>Multichannel pipette (0.5 - 10 &#181;l)</td>
			<td>Gilson</td>
			<td>FA10013</td>
			<td></td>
		</tr>
		<tr>
			<td>Multichannel pipette (20 - 200 &#181;l)</td>
			<td>Gilson</td>
			<td>FA10011</td>
			<td></td>
		</tr>
		<tr>
			<td>Mycobacterium bovis BCG&#160;</td>
			<td>American Type Culture Collection</td>
			<td>ATCC35734</td>
			<td>strain TMC 1011 [BCG Pasteur]</td>
		</tr>
		<tr>
			<td>OADC enrichment</td>
			<td>BD</td>
			<td>211886</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>NZYTech</td>
			<td>MB25201</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 medium</td>
			<td>Gibco</td>
			<td>21875091</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Gibco</td>
			<td>11360-070</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrophotometer UV-6300PC</td>
			<td>VWR</td>
			<td>634-6041</td>
			<td></td>
		</tr>
		<tr>
			<td>Square Petri dish 120 x 120 mm</td>
			<td>Corning</td>
			<td>BP124-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Tyloxapol</td>
			<td>Sigma-Aldrich</td>
			<td>T8761</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasound bath Elma P 30 H</td>
			<td>VWR</td>
			<td>142-0051</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

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

The mCFU assay described here increases the amount of information that can be retrieved from a single Petri dish to at least 96-fold. Figure 5&#160;depicts a comparison of two drug-delivery methods for the repurposed use of saquinavir (SQV)31,32&#160;as a host-directed drug to treat tuberculosis. In this assay, four different strains of Mycobacterium tuberculosis were used to infect primary human macrophages. M. tubercul...

Watch this Scientific Journal Video about Optimizing CFU Determination for Efficient Assessment of TB Vaccine Efficacy and Antigen Presentation Analysis at JoVE.com

Optimizing CFU Determination for Efficient Assessment of TB Vaccine Efficacy and Antigen Presentation Analysis (Video) | JoVE

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 ...