Name: opsonophagocytic killing assay to assess immunological responses against bacterial pathogens
Uploaded: 2019-04-05T14:00:00.000+00:00
Duration: 8 min 47 s
Description: Watch this Scientific Journal Video about Opsonophagocytic Killing Assay to Assess Immunological Responses Against Bacterial Pathogens at JoVE.com

We thank Dr. Moon Nahm (University of Alabama Birmingham) for his invaluable assistance in establishing OPKA assays in our laboratory. This work was supported by National Institutes of Health Grant 1R01AI123383-01A1 to FYA.

1. Culture, Differentiation, and Validation of HL-60 Cells

<ol>
	<li>Prepare HL-60 cell culture media composed of 500 mL RPMI with L-glutamine and 50 mL heat-inactivated fetal bovine serum. Do not add antibiotics as this may affect the differentiation of the HL-60 cells.</li>
	<li>For propagation/maintenance of HL-60 cells, culture 5 x 106 cells in 10 mL of HL-60 cell culture media in 75 cm2 vented flasks at 37 &#176;C and 5% CO2. Passage cells every 3&#8722;4 days to maintain optimal cell concentrations. 
	NOTE: The cell concentration should not exceed 5 x 106/mL.</li>
	<li>Generate working stocks of HL-60 cells by aliquoting approximately 1 x 106 cells/mL in HL60 culture media with 10% dimethyl sulfoxide (DMSO) into 1 mL cryogenic tubes. 
	NOTE: Working stocks may be stored at -80 &#176;C. The master stock should be stored at -120 &#176;C.</li>
	<li>Differentiate HL-60 cells by culturing 1.5 x 107 cells in 15 mL of HL-60 cell culture media with 0.6%&#160;N,N-dimethylformamide (DMF) at 37 &#176;C and 5% CO2 in sterile filter-capped 75 cm2 flasks for 3 days prior to OPKA.</li>
	<li>Validate that the HL-60 cells have been successfully differentiated and are appropriate for use in the OPKA assay by testing viability and cell surface markers according to established flow cytometry protocols16,17,18,19. After differentiation, harvest HL-60 cells and stain approximately 1 x 104 cells with fluorescently-conjugated antibodies/stains for CD71, CD35, annexin V, and propidium iodide. 
	NOTE: Differentiated cells should be &#8805;65% viable, &#8805;55% CD35+, and &#8804;20% CD71+ as determined through established validation protocols14 (Figure 1).</li>
</ol>

2. Preparation of OPKA Buffers and Reagents

<ol>
	<li>Prepare 50 mL of sterile opsonization buffer B (OBB) by mixing 42.5 mL of sterile 1x phosphate-buffered saline (PBS) with Ca2+/Mg2+, 5 mL of heat-inactivated fetal bovine serum, and 2.5 mL of 0.1% sterile gelatin. Store at 4 &#176;C.</li>
	<li>Obtain baby rabbit complement and store at -80 &#176;C.</li>
	<li>Obtain or prepare bacterial culture plates (i.e., 15 x 100 mm2 5% sheep&#8217;s blood agar plates).</li>
</ol>

3. Preparation of Bacterial Stock Samples

<ol>
	<li>Obtain a stock of the bacterial strain(s) to be tested. 
	NOTE: For this protocol, serotype 3 Streptococcus pneumoniae (WU2, generously provided by Dr. Moon Nahm) is used.</li>
	<li>Grow the bacterial strain in an appropriate broth (i.e., Todd-Hewitt broth + 0.5% yeast extract for this WU2 strain) for approximately 2&#8722;4 h at 37 &#176;C. 
	NOTE: The optical density at 600 nm (OD600) of the culture should be between 0.6 and 0.8.</li>
	<li>Pellet the bacteria by centrifugation at 6,000 x g for 2 min and resuspend the cells in 10&#8722;30 mL of 15% glycerol in the appropriate broth. Aliquot the bacterial culture (500 &#181;L per aliquot) into sterile 1.5 mL centrifuge tubes and store at -80 &#176;C.</li>
	<li>Thaw out one vial of bacterial stock in a 37 &#176;C water bath. Pellet the bacterial cells and resuspend in 500 &#181;L of OBB under sterile conditions.</li>
	<li>Prepare different dilutions of the bacterial stock in OBB (i.e., 10 &#181;L of no dilution, 10 &#181;L of 1:10, 10 &#181;L of 1:100, etc.). Perform the OPKA assay (sections 4&#8211;6, including HL-60/complement co-culture) as described below using various dilutions of the untreated bacterial stock. Culture the plates overnight at 30 &#176;C (no CO2). 
	NOTE: The temperature 30 &#176;C is specific for WU2 to prevent overgrowth; other strains/serotypes may grow optimally at 37 &#176;C.</li>
	<li>Count the colonies for each dilution of untreated bacterial stock co-cultured with HL-60 cells and complement. Determine which dilution of bacteria yields the optimal number of countable colonies (approximately 80&#8722;120 CFUs for untreated bacteria co-cultured with HL-60 cells). Note this dilution for future OPKAs involving this bacterial stock.</li>
</ol>

4. Bacterial Treatment and Culture

<ol>
	<li>Thaw one tube of bacterial stock prepared in step 3.3. Pellet bacteria (6,000 x g for 2 min) and resuspend cell pellet in OBB at optimal dilution as determined in step 3.6.</li>
	<li>Pipette 10 &#181;L of resuspended bacterial dilution per well in a round-bottom 96-well cell culture plate.</li>
	<li>Add 20 &#181;L of appropriate antibody or drug treatment to each experimental well in duplicate. 
	NOTE: In this protocol, a serotype-specific antibody generated in mice is added as treatment X and a glycoside hydrolase enzyme known to degrade the serotype 3 polysaccharide capsule is added as treatment Y (Figure 2)4,20. For control wells, use 1x PBS or OBB, depending on the buffer used for treatment wells.</li>
	<li>Shake the sample plate at approximately 90 rpm for 1 h at room temperature. Adjust these conditions depending on the optimal temperature or shaking conditions of the treatments being tested.</li>
</ol>

5. HL-60 bacterial Co-culture

<ol>
	<li>Prepare HL-60 cells by harvesting the HL-60 differentiated cells that are treated with DMF three days prior (see step 1.4) into 15 mL conical tubes. Pellet the cells (500 x g, 3 min), discard the supernatant, and wash with at least 10 mL of 1x PBS.</li>
	<li>Pellet the washed cells (500 x g, 3 min), discard the supernatant, and resuspend the cells in OBB (start with 1 mL OBB and adjust for a final concentration of 1 x 107/mL after cell counting).</li>
	<li>Add baby rabbit complement (sterile, undiluted baby rabbit serum, age 3&#8722;4 weeks) at a 1:5 final volume. 
	NOTE: The final concentration of the HL-60-complement mixture should be 1 x 107/mL. If testing complement dependency, a second solution containing active HL-60 cells with heat-inactivated complement may be used (complement may be inactivated by incubating in a water bath at &#62;55 &#176;C for at least 30 min).</li>
	<li>After 1 h bacterial culture is complete (step 4.4), divide each sample (i.e., 10 &#181;L of each 30 &#181;L sample well into two new wells) into duplicate wells for two groups (i.e., use only 20 &#181;L of the original 30 &#181;L co-culture to account for pipetting error): one set will be co-cultured with HL-60-complement and one will include bacteria only. Add 50 &#181;L of the HL-60-complement mixture (from step 5.3) to each experimental set of wells (delegated +HL-60); add 50 &#181;L of OBB alone to the wells of bacteria only (delegated -HL-60). 
	NOTE: For this example, approximately 800 bacterial CFUs are used for the initial co-culture with 5 x 105/50 &#181;L HL-60 cells. If this multiplicity of infection is too high or too low as indicated by final colony numbers, adjust the initial bacterial dilution as opposed to the HL-60 cell count.</li>
	<li>Shake the 96-well plate at 37 &#176;C for 1 h (no CO2).</li>
</ol>

6. Sample Plating and Overnight Incubation

<ol>
	<li>Dilute each well 1:5 with OBB, so that each sample has a volume of at least 50 &#181;L.</li>
	<li>Pipette 50 &#181;L of each sample directly onto a designated area of a bacterial culture plate, ensuring adequate spacing between samples. For 15 x 100 mm2 round agar plates, pipet approximately 4 samples onto one plate.</li>
	<li>Cover and allow samples to dry for approximately 15 min at room temperature.</li>
	<li>Invert plates and culture overnight at 30 &#176;C (no CO2). Alternatively, culture plates in anaerobic jars to test whether anoxic conditions affect the bacterial growth or to control for morphology.</li>
	<li>After overnight culture, count the colonies in each designated sample area. Analyze data by comparing the number of live cells in each set to the corresponding control and/or samples that do not receive HL-60 cell co-culture (indicative of 100% cell survival, 0% cell killing).</li>
</ol>

<ol>
	<li>Romero-Steiner, S., 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=Standardization+of+an+opsonophagocytic+assay+for+the+measurement+of+functional+antibody+activity+against+Streptococcus+pneumoniae+using+differentiated+HL-60+cells.">Standardization of an opsonophagocytic assay for the measurement of functional antibody activity against Streptococcus pneumoniae using differentiated HL-60 cells.</a> Clinical and Diagnostic Laboratory Immunology. 4 (4), 415-422 (1997).</li><li>Nanra, J. S., 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=Capsular+polysaccharides+are+an+important+immune+evasion+mechanism+for+Staphylococcus+aureus.">Capsular polysaccharides are an important immune evasion mechanism for Staphylococcus aureus.</a> Human Vaccines and Immunotherapeutics. 9 (3), 480-487 (2013).</li><li>Ishibashi, K., Yamaguchi, O., Shiraiwa, Y., Ogihara, M., Shigeta, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combination+therapy+of+Pseudomonas+aeruginosa+pyelonephritis+in+neutropenic+mice+with+human+antilipopolysaccharide+monoclonal+antibody+and+cefsulodin.">Combination therapy of Pseudomonas aeruginosa pyelonephritis in neutropenic mice with human antilipopolysaccharide monoclonal antibody and cefsulodin.</a> Journal of Urology. 155 (6), 2094-2097 (1996).</li><li>Middleton, D. R., Paschall, A. V., Duke, J. A., Avci, F. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymatic+Hydrolysis+of+Pneumococcal+Capsular+Polysaccharide+Renders+the+Bacterium+Vulnerable+to+Host+Defense.">Enzymatic Hydrolysis of Pneumococcal Capsular Polysaccharide Renders the Bacterium Vulnerable to Host Defense.</a> Infection and Immunity. , (2018).</li><li>Baker, C. J., Noya, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potential+use+of+intravenous+immune+globulin+for+group+B+streptococcal+infection.">Potential use of intravenous immune globulin for group B streptococcal infection.</a> Reviews of Infectious Diseases. 12, 476-482 (1990).</li><li>Tian, H., Weber, S., Thorkildson, P., Kozel, T. R., Pirofski, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficacy+of+opsonic+and+nonopsonic+serotype+3+pneumococcal+capsular+polysaccharide-specific+monoclonal+antibodies+against+intranasal+challenge+with+Streptococcus+pneumoniae+in+mice.">Efficacy of opsonic and nonopsonic serotype 3 pneumococcal capsular polysaccharide-specific monoclonal antibodies against intranasal challenge with Streptococcus pneumoniae in mice.</a> Infection and Immunity. 77 (4), 1502-1513 (2009).</li><li>Parameswarappa, S. 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=A+Semi-synthetic+Oligosaccharide+Conjugate+Vaccine+Candidate+Confers+Protection+against+Streptococcus+pneumoniae+Serotype+3+Infection.">A Semi-synthetic Oligosaccharide Conjugate Vaccine Candidate Confers Protection against Streptococcus pneumoniae Serotype 3 Infection.</a> Cell Chemical Biology. 23 (11), 1407-1416 (2016).</li><li>Jackson, L., 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=Randomized+clinical+trial+of+a+single+versus+a+double+dose+of+13-valent+pneumococcal+conjugate+vaccine+in+adults+55+through+74+years+of+age+previously+vaccinated+with+23-valent+pneumococcal+polysaccharide+vaccine.">Randomized clinical trial of a single versus a double dose of 13-valent pneumococcal conjugate vaccine in adults 55 through 74 years of age previously vaccinated with 23-valent pneumococcal polysaccharide vaccine.</a> Vaccine. 36 (5), 606-614 (2018).</li><li>Cywes-Bentley, C., 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=Antibody+to+a+conserved+antigenic+target+is+protective+against+diverse+prokaryotic+and+eukaryotic+pathogens.">Antibody to a conserved antigenic target is protective against diverse prokaryotic and eukaryotic pathogens.</a> Proceedings of the National Academy of Sciences of the USA. 110 (24), 2209-2218 (2013).</li><li>Fleck, R. A., Romero-Steiner, S., Nahm, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+HL-60+cell+line+to+measure+opsonic+capacity+of+pneumococcal+antibodies.">Use of HL-60 cell line to measure opsonic capacity of pneumococcal antibodies.</a> Clinical and Diagnostic Laboratory Immunology. 12 (1), 19-27 (2005).</li><li>Collins, S. J., Ruscetti, F. W., Gallagher, R. E., Gallo, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Terminal+differentiation+of+human+promyelocytic+leukemia+cells+induced+by+dimethyl+sulfoxide+and+other+polar+compounds.">Terminal differentiation of human promyelocytic leukemia cells induced by dimethyl sulfoxide and other polar compounds.</a> Proceedings of the National Academy of Sciences of the USA. 75 (5), 2458-2462 (1978).</li><li>Melnick, N., Rajam, G., Carlone, G. M., Sampson, J. S., Ades, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+a+novel+therapeutic+approach+to+treating+severe+pneumococcal+infection+using+a+mouse+model.">Evaluation of a novel therapeutic approach to treating severe pneumococcal infection using a mouse model.</a> Clinical and Vaccine Immunology. 16 (6), 806-810 (2009).</li><li>Dwyer, M., Gadjeva, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Opsonophagocytic+assay.">Opsonophagocytic assay.</a> Methods in Molecular Biology. 1100, 373-379 (2014).</li><li>Burton, R. L., Nahm, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+fourfold+multiplexed+opsonophagocytosis+assay+for+pneumococcal+antibodies+against+additional+serotypes+and+discovery+of+serological+subtypes+in+Streptococcus+pneumoniae+serotype+20.">Development of a fourfold multiplexed opsonophagocytosis assay for pneumococcal antibodies against additional serotypes and discovery of serological subtypes in Streptococcus pneumoniae serotype 20.</a> Clinical and Vaccine Immunololgy. 19 (6), 835-841 (2012).</li><li>Clarke, M. L., 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=Low-cost,+high-throughput,+automated+counting+of+bacterial+colonies.">Low-cost, high-throughput, automated counting of bacterial colonies.</a> Cytometry Part A. 77 (8), 790-797 (2010).</li><li>Aanei, C. M., 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=Database-guided+Flow-cytometry+for+Evaluation+of+Bone+Marrow+Myeloid+Cell+Maturation.">Database-guided Flow-cytometry for Evaluation of Bone Marrow Myeloid Cell Maturation.</a> Journal of Visualized Experiments. (141), e57867 (2018).</li><li>Hirz, T., Dumontet, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+Isolation+and+Analysis+to+Determine+their+Role+in+Lymphoma+Cell+Sensitivity+to+Therapeutic+Agents.">Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents.</a> Journal of Visulaized Experiments. (109), e53846 (2016).</li><li>Rieger, A. M., Nelson, K. L., Konowalchuk, J. D., Barreda, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modified+annexin+V/propidium+iodide+apoptosis+assay+for+accurate+assessment+of+cell+death.">Modified annexin V/propidium iodide apoptosis assay for accurate assessment of cell death.</a> Journal of Visualized Experiments. (50), 2597 (2011).</li><li>Blair, O. C., Carbone, R., Sartorelli, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+HL-60+promyelocytic+leukemia+cells:+simultaneous+determination+of+phagocytic+activity+and+cell+cycle+distribution+by+flow+cytometry.">Differentiation of HL-60 promyelocytic leukemia cells: simultaneous determination of phagocytic activity and cell cycle distribution by flow cytometry.</a> Cytometry. 7 (2), 171-177 (1986).</li><li>Middleton, D. 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=Identification+and+characterization+of+the+Streptococcus+pneumoniae+type+3+capsule-specific+glycoside+hydrolase+of+Paenibacillus+species+32352.">Identification and characterization of the Streptococcus pneumoniae type 3 capsule-specific glycoside hydrolase of Paenibacillus species 32352.</a> Glycobiology. 28 (2), 90-99 (2018).</li></ol>

The authors have nothing to disclose.

OPKAs serve essential roles in assessing antibody mediated immune responses induced by vaccinations6,8. The main significance of this simplified OPKA is the adaptability in the conditions to be tested (i.e., antibodies, enzyme treatments, etc.). In this sense, while this assay can be used to test the contribution of opsonins (i.e., antibodies) in phagocytosis, it can also be used to assess ways to overcome virulence factors (i.e., capsular polysaccharides) that n...

The opsonophagocytic killing assay (OPKA) is a critical tool for linking alterations in bacterial structure or function to subsequent changes in immune response and function. As such, it is frequently used as a complementary assay to determine immune-based efficacy of antibody treatments, vaccine candidates, enzyme optimization, etc. While in vivo assays are necessary to determine effective clearance or protection in a bacterial infection model, the OPKA can be used to assess immune contribution to bacterial cell death at the most basic components: bacteria, immune cells, and experimental treatments. Previous studies have shown that OPKAs can be modified and used for a variety of bacteria and serotypes, including Streptococcus pneumoniae1, Staphylococcus aureus2, Pseudomonas aeruginosa3. Furthermore, these optimized assays can be used to assess different experimental treatments, including the ability of an enzyme to make the bacterium more accessible to complement-mediated immune cells4 and antibody treatments to improve opsonization5. Classically, OPKA assay has been successfully used in basic and clinical research settings as a powerful indicator for protection induced by pathogen-specific antibodies6,7,8,9.
Different types of immune cells may be used for assessment of opsonophagocytic killing. One commonly used phagocytic population is the HL-60 human leukemic cell line. This cell line can be kept as inactivated promyelocytes in culture; however, they can be differentiated into various activated states via different drug treatments10,11. Treatment of HL60 with N,N-dimethylformamide differentiates the cell line into activated neutrophils with strong phagocytic activity11. While HL-60 cells have been optimized and are frequently used for these phagocytosis assays10, other primary polymorphonuclear leukocytes can be used as the immune arm of the experiment12.
Additionally, these assays can be simplified13 or multiplexed14 to look at multiple antibiotic-resistant strains of the bacteria to be tested. The multiplexed method has been made more feasible through the development of software that can efficiently count bacterial colony forming units (CFUs) per spot on an agar plate15. Here, we describe a streamlined method using one bacterial strain, HL-60 cells, baby rabbit complement, and blood agar plates. With this method, multiple treatments can be assessed quickly to address specific research questions on how the innate immune response to bacterial infection can be modulated.

<table><tbody><tr><td>Annexin V (APC conjugated)</td><td>BioLegend</td><td>640919</td><td></td></tr><tr><td>anti-CD35, human (PE conjugated)</td><td>BioLegend</td><td>333405</td><td></td></tr><tr><td>anti-CD71, human (PE conjugated)</td><td>BioLegend</td><td>334105</td><td></td></tr><tr><td>bacterial strain to be used (i.e., &lt;em&gt;Streptococcus pneumoniae&lt;/em&gt;, WU2)</td><td>Bacterial Respiratory Reference Laboratory (Dr. Moon Nahm)&amp;nbsp;</td><td></td><td></td></tr><tr><td>blood agar plates</td><td>Hardy Diagnostic</td><td>A10</td><td></td></tr><tr><td>Fetal Clone serum</td><td>HyClone</td><td>SH30080.03</td><td></td></tr><tr><td>glycerol</td><td>Sigma</td><td>G9012-1L</td><td></td></tr><tr><td>HL-60 cells</td><td>ATCC</td><td>CCL-240</td><td></td></tr><tr><td>IgG Isotype Control (PE conjugated)</td><td>BioLegend</td><td>400907</td><td></td></tr><tr><td>N,N-dimethylformamide (DMF)</td><td>Fisher Chemical</td><td>UN2265</td><td></td></tr><tr><td>propidium iodide</td><td>Sigma</td><td>P4864</td><td></td></tr><tr><td>RPMI media with L-glutamine</td><td>Corning</td><td>10-040-CV</td><td></td></tr></tbody></table>

opsonophagocytic killing assay to assess immunological responses against bacterial pathogens

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental procedure in which phagocytic cells are co-cultured with bacterial units. The immune cells will phagocytose and kill the bacterial cultures in a complement-dependent manner. The efficiency of the immune-mediated cell killing is dependent on a number of factors and can be used to determine how different bacterial cultures compare with regard to resistance to cell death. In this way, the efficacy of potential immune-based therapeutics can be assessed against specific bacterial strains and/or serotypes. In this protocol, we describe a simplified OPKA that utilizes basic culture conditions and cell counting to determine bacterial cell viability after co-culture with treatment conditions and HL-60 immune cells. This method has been successfully utilized with a number of different pneumococcal serotypes, capsular and acapsular strains, and other bacterial species. The advantages of this OPKA protocol are its simplicity, versatility (as this assay is not limited to antibody treatments as opsonins), and minimization of time and reagents to assess basic experimental groups.

Validation of HL-60 differentiation should be performed before starting the OPKA. This can be accomplished using flow cytometry to determine the extracellular expression of CD11b, CD35, CD71, and annexin V (Figure 1). Propidium iodide can also be used as a viability marker. After being treated with DMF for 3 days, expression of CD35 should be increased (&#8805;55% of all cells) and expression of CD71 should be decreased (&#8804;20% of all cells). The percentage of annexin V+ and propidium io...

Watch this Scientific Journal Video about Opsonophagocytic Killing Assay to Assess Immunological Responses Against Bacterial Pathogens at JoVE.com

Opsonophagocytic Killing Assay to Assess Immunological Responses Against Bacterial Pathogens

This opsonophagocytic killing assay is used to compare the ability of phagocytic immune cells to respond to and kill bacteria based on different treatments and/or conditions. Classically, this assay serves as the gold&#160;standard for assessing effector functions of antibodies raised against a bacterium as opsonin.

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental ...

opsonophagocytic-killing-assay-to-assess-immunological-responses

Research

JoVE Journal

Immunology and Infection

12.1K Views.  University of Georgia. This opsonophagocytic killing assay is used to compare the ability of phagocytic immune cells to respond to and kill bacteria based on different treatments and/or conditions. Classically, this assay serves as the gold standard for assessing effector functions of antibodies raised against a bacterium as opsonin.

Video: Opsonophagocytic Killing Assay to Assess Immunological Responses Against Bacterial Pathogens

Determining the Phagocytic Activity of Clinical Antibody Samples

Antibody-driven phagocytosis is induced via the engagement of Fc receptors on professional phagocytes, and can contribute to both clearance as well as pathology of disease. While the properties of the variable domains of antibodies have long been considered critical to in vivo function, the ability of antibodies to recruit innate immune cells via their Fc domains has become increasingly appreciated as a major factor in their efficacy, both in the setting of recombinant monoclonal antibody therapy, as well as in the course of natural infection or vaccination1-3.
Importantly, despite its nomenclature as a constant domain, the antibody Fc domain does not have constant function, and is strongly modulated by IgG subclass (IgG1-4) and glycosylation at Asparagine 2974-6. Thus, this method to study functional differences of antigen-specific antibodies in clinical samples will facilitate correlation of the phagocytic potential of antibodies to disease state, susceptibility to infection, progression, or clinical outcome.
Furthermore, this effector function is particularly important in light of the documented ability of antibodies to enhance infection by providing pathogens access into host cells via Fc receptor-driven phagocytosis7. Additionally, there is some evidence that phagocytic uptake of immune complexes can impact the Th1/Th2 polarization of the immune response8.
Here, we describe an assay designed to detect differences in antibody-induced phagocytosis, which may be caused by differential IgG subclass, glycan structure at Asn297, as well as the ability to form immune complexes of antigen-specific antibodies in a high-throughput fashion. To this end, 1 &mu;m fluorescent beads are coated with antigen, then incubated with clinical antibody samples, generating fluorescent antigen specific immune complexes. These antibody-opsonized beads are then incubated with a monocytic cell line expressing multiple Fc&gamma;Rs, including both inhibitory and activating. Assay output can include phagocytic activity, cytokine secretion, and patterns of Fc&gamma;Rs usage, and are determined in a standardized manner, making this a highly useful system for parsing differences in this antibody-dependent effector function in both infection and vaccine-mediated protection9.

We present a high-throughput flow cytometric assay to determine the phagocytic activity of antigen-specific antibodies from clinical samples, utilizing fluorescent antigen-coated beads and a monocytic cell line expressing multiple Fc receptors&#x2014;providing receptor usage and phagocytic activity determinations in a standardized and reproducible fashion for any antigen of interest.

Antibody-driven phagocytosis is induced via the engagement of Fc receptors on professional phagocytes, and can contribute to both clearance as well as ...

A Visual Assay to Monitor T6SS-mediated Bacterial Competition

Type VI secretion systems (T6SSs) are molecular nanomachines allowing Gram-negative bacteria to transport and inject proteins into a wide variety of target cells1,2. The T6SS is composed of 13 core components and displays structural similarities with the tail-tube of bacteriophages3. The phage uses a tube and a puncturing device to penetrate the cell envelope of target bacteria and inject DNA. It is proposed that the T6SS is an inverted bacteriophage device creating a specific path in the bacterial cell envelope to drive effectors and toxins to the surface. The process could be taken further and the T6SS device could perforate other cells with which the bacterium is in contact, thus injecting the effectors into these targets. The tail tube and puncturing device parts of the T6SS are made with Hcp and VgrG proteins, respectively4,5.
The versatility of the T6SS has been demonstrated through studies using various bacterial pathogens. The Vibrio cholerae T6SS can remodel the cytoskeleton of eukaryotic host cells by injecting an "evolved" VgrG carrying a C-terminal actin cross-linking domain6,7. Another striking example was recently documented using Pseudomonas aeruginosa which is able to target and kill bacteria in a T6SS-dependent manner, therefore promoting the establishment of bacteria in specific microbial niches and competitive environment8,9,10.
In the latter case, three T6SS-secreted proteins, namely Tse1, Tse2 and Tse3 have been identified as the toxins injected in the target bacteria (Figure 1). The donor cell is protected from the deleterious effect of these effectors via an anti-toxin mechanism, mediated by the Tsi1, Tsi2 and Tsi3 immunity proteins8,9,10. This antimicrobial activity can be monitored when T6SS-proficient bacteria are co-cultivated on solid surfaces in competition with other bacterial species or with T6SS-inactive bacteria of the same species8,11,12,13.
The data available emphasized a numerical approach to the bacterial competition assay, including time-consuming CFU counting that depends greatly on antibiotic makers. In the case of antibiotic resistant strains like P. aeruginosa, these methods can be inappropriate. Moreover, with the identification of about 200 different T6SS loci in more than 100 bacterial genomes14, a convenient screening tool is highly desirable. We developed an assay that is easy to use and requires standard laboratory material and reagents. The method offers a rapid and qualitative technique to monitor the T6SS-dependent bactericidal/bacteriostasis activity by using a reporter strain as a prey (in this case Escherichia coli DH5&alpha;) allowing a-complementation of the lacZ gene. Overall, this method is graphic and allows rapid identification of T6SS-related phenotypes on agar plates. This experimental protocol may be adapted to other strains or bacterial species taking into account specific conditions such as growth media, temperature or time of contact.

We describe a qualitative assay to monitor bacterial competition mediated by the Pseudomonas aeruginosa type VI secretion system (T6SS). The assay relies on the survival/killing of Escherichia coli target cells carrying a lacZ-reporter. This technique is adjustable to assess the bactericidal/bacteriostasis activity of T6SS-proficient microorganisms.

Type VI secretion systems (T6SSs) are molecular  nanomachines allowing Gram-negative bacteria to transport and inject proteins  into a wide variety of ...

A Rapid Image-based Bacterial Virulence Assay Using Amoeba

Traditional bacterial virulence assays involve prolonged exposure of bacteria over the course of several hours to host cells. During this time, bacteria can undergo changes in the physiology due to the exposure to host growth environment and the presence of the host cells. We developed an assay to rapidly measure the virulence state of bacteria that minimize the extent to which bacteria grow in the presence of host cells. Bacteria and amoebae are mixed together and immobilized on a single imaging plane using an agar pad. The procedure uses single-cell fluorescence imaging with calcein-acetoxymethyl ester (calcein-AM) as an indicator of host cell health. The fluorescence of host cells is analyzed after 1 h of exposure of host cells to bacteria using epifluorescence microscopy. Image analysis software is used to compute a host killing index. This method has been used to measure virulence within planktonic and surface-attached Pseudomonas aeruginosa sub-populations during the initial stage of biofilm formation and may be adapted to other bacteria and other stages of biofilm growth. This protocol provides a rapid and robust method of measuring virulence and avoids many of the complexities associated with the growth and maintenance of mammalian cell lines. Virulence phenotypes measured here using amoebae have also been validated using mouse macrophages. In particular, this assay was used to establish that surface attachment upregulates virulence in P. aeruginosa.

Here, we present a protocol to measure the virulence of planktonic or surface-attached bacteria using D. discoideum (amoeba) as a host. Virulence is measured over a period of 1 h and host killing is quantified using fluorescence microscopy and image analysis. We demonstrate this protocol using the bacterium P. aeruginosa.

Traditional bacterial virulence assays involve prolonged exposure of bacteria over the course of several hours to host cells. During this time, bacteria ...

Bioengineering

Evaluation of Host-Pathogen Responses and Vaccine Efficacy in Mice

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a striking increase in life expectancy around the globe. Nonetheless, determining vaccine efficacy remains a challenge. Emerging evidence suggests that the current acellular vaccine (aPV) for Bordetella pertussis (B. pertussis) induces suboptimal immunity. Therefore, a major challenge is designing a next-generation vaccine that induces protective immunity without the adverse side effects of a whole-cell vaccine (wPV). Here we describe a protocol that we used to test the efficacy of a promising, novel adjuvant that skews immune responses to a protective Th1/Th17 phenotype and promotes a better clearance of a B. pertussis challenge from the murine respiratory tract. This article describes the protocol for mouse immunization, bacterial inoculation, tissue harvesting, and analysis of immune responses. Using this method, within our model, we have successfully elucidated crucial mechanisms elicited by a promising, next-generation acellular pertussis vaccine. This method can be applied to any infectious disease model in order to determine vaccine efficacy.

Here we present an elegant protocol for in vivo evaluation of vaccine effectiveness and host immune responses. This protocol can be adapted for vaccine models that study viral, bacterial, or parasitic pathogens.

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a ...

A High-throughput Shigella-specific Bactericidal Assay

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing activity by assessing the ability of antibodies in serum to bind to bacteria and activate complement. This complement activation results in the lysis and killing of the target bacteria. These assays are valuable because they go beyond quantifying antibody production to elucidate the biological functions that these antibodies have, allowing researchers to study the role that antibodies may play in preventing infection. SBAs have been used to study immune responses for many human pathogens, but there is no widely accepted methodology for Shigella at present. Historically, SBAs have been very labor-intensive, requiring many time-consuming steps to accurately quantify surviving bacteria. This protocol describes a simple, robust, and high-throughput assay that measures functional antibodies specific for Shigella in serum in vitro. The method described here offers many advantages over traditional SBAs, including the use of frozen bacterial stocks, 96 well assay plates, a micro-culture system, and automated colony-counting. All of these modifications make this assay less labor-intensive and more high-throughput. This protocol is simpler and faster to perform than traditional SBAs while still using simple technologies and readily available reagents. The protocol has been successfully applied in multiple independent laboratories and the assay is robust and reproducible. The assay can be used to assess immune responses in pre-clinical as well as clinical studies. Quantifying shigellacidal antibody titers both before and after antigen exposure (either by immunization or infection) allows for a broader understanding of how functional antibody responses are generated and their contribution to protective immunity. The development of this standardized, well-characterized assay may greatly facilitate Shigella vaccine design.

A High-throughput Shigella-specific Bactericidal Assay

Here we present a protocol to measure Shigellacidal activity of antibodies in serum. Serum is mixed with bacteria and exogenous complement, incubated, and the reaction mixture is plated on agar plates. Viable bacteria form colonies which are counted, using an automated colony enumerator, and used to determine the bactericidal titer.

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing ...

Opsono-Adherence Assay to Evaluate Functional Antibodies in Vaccine Development Against Bacillus anthracis and Other Encapsulated Pathogens

The opsono-adherence assay is a functional assay that enumerates the attachment of bacterial pathogens to professional phagocytes. Because adherence is requisite to phagocytosis and killing, the assay is an alternative method to&#160;opsono-phagocytic killing assays. An advantage of the opsono-adherence&#160;assay is the option of using inactivated pathogens and mammalian cell lines, which allows standardization across multiple experiments. The use of an inactivated pathogen in the assay also facilitates work with biosafety level 3 infectious agents and other virulent pathogens. In our work, the opsono-adherence assay was used to assess the functional ability of antibodies, from sera of animals immunized with an anthrax capsule-based vaccine, to induce adherence of fixed Bacillus anthracis to a mouse macrophage cell line, RAW 264.7. Automated fluorescence microscopy was used to capture images of bacilli adhering to macrophages. Increased adherence was correlated with the presence of anti-capsule antibodies in the serum. Non-human primates that exhibited high serum anti-capsule antibody concentrations were protected from anthrax challenge. Thus, the opsono-adherence assay can be used to elucidate the biological functions of antigen specific antibodies in sera, to evaluate the efficacy of vaccine candidates and other therapeutics, and to serve as a possible correlate of immunity.&#160;&#160;&#160;&#160;&#160;&#160;

Opsono-Adherence Assay to Evaluate Functional Antibodies in Vaccine Development Against Bacillus anthracis and Other Encapsulated Pathogens

The opsono-adherence assay is an alternative method to the opsono-phagocytic killing assay to evaluate the opsonic functions of antibodies in vaccine development.

The opsono-adherence assay is a functional assay that enumerates the attachment of bacterial pathogens to professional phagocytes. Because adherence is ...

A Serum Bactericidal Assay for the Complement-Mediated Bactericidal Activity of Antibodies

Source: Weerts, H. P., et al. <a href="https://app.jove.com/v/59164">A High-throughput Shigella-specific Bactericidal Assay.</a> J. Vis. Exp. (2019).
This video demonstrates a technique to evaluate the complement-mediated bactericidal activity of antibodies present in serum. This approach combines isolated serum with pathogenic bacteria and exogenous complement proteins. Upon incubation, the bacteria are grown on an agar plate, and the produced colonies are quantified to measure the serum bactericidal titer.

Source: Weerts, H. P., et al. A High-throughput Shigella-specific Bactericidal Assay. J. Vis. Exp. (2019).
This video demonstrates a technique to evaluate ...

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Specialized Assays

An Assay for Bacterial Extracellular Protein-Mediated Cytotoxicity on PMNs

Source: Dankoff, J. G., et al. <a href="https://app.jove.com/v/60681">Quantifying the Cytotoxicity of Staphylococcus aureus Against Human Polymorphonuclear Leukocytes.</a> J. Vis. Exp. (2020).
This video demonstrates an assay to investigate the cytotoxicity of proteins produced by Staphylococcus aureus, a pathogenic bacterium, on isolated human polymorphonuclear leukocytes (PMNs). The secreted cytotoxic proteins form pores on the PMN cell membrane, initiating cell death. Upon staining the dead cells using propidium iodide, flow cytometry is performed to quantify the dead cells and assess the cytotoxicity of the bacteria.

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human ...

Cytotoxicity Assay of Staphylococcus aureus Strains on Neutrophils following Phagocytosis

Source: Dankoff, J. G. et al., <a href="https://app.jove.com/v/60681">Quantifying the Cytotoxicity of Staphylococcus aureus Against Human Polymorphonuclear Leukocytes.</a> J. Vis. Exp. (2020)
In this video, we demonstrate an assay to detect the cytotoxicity of virulent and less virulent Staphylococcus aureus strains against human neutrophils following phagocytosis using flow cytometry.

1.&#160;S. aureus cytotoxicity assay against human polymorphonuclear leukocytes following phagocytosis
NOTE: Growth curves defined by the optical density ...

Assessment of Opsonophagocytic Killing Activity against Bacterial Pathogens

Source: Paschall, A. V. et al., <a href="https://app.jove.com/v/59400">Opsonophagocytic Killing Assay to Assess Immunological Responses Against Bacterial Pathogens.</a>&#160;J. Vis. Exp.&#160;(2019)
The video shows an in vitro opsonophagocytic killing assay using a co-culture of human neutrophils with Streptococcus pneumoniae. Antibodies and complement proteins facilitate opsonization of the bacteria, enabling neutrophils to recognize and kill bacteria, showing successful neutrophil opsonophagocytic activity.

1. Culture, Differentiation, and Validation of HL-60 Cells

	Prepare HL-60 cell culture media composed of 500 mL Roswell Park Memorial Institute (RPMI) ...

Opsonophagocytic Killing Assay to Assess Immunological Responses Against Bacterial Pathogens

Summary

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Chapters in this video

Determining the Phagocytic Activity of Clinical Antibody Samples

A Visual Assay to Monitor T6SS-mediated Bacterial Competition

A Rapid Image-based Bacterial Virulence Assay Using Amoeba

Evaluation of Host-Pathogen Responses and Vaccine Efficacy in Mice

A High-throughput Shigella-specific Bactericidal Assay

Opsono-Adherence Assay to Evaluate Functional Antibodies in Vaccine Development Against Bacillus anthracis and Other Encapsulated Pathogens

A Serum Bactericidal Assay for the Complement-Mediated Bactericidal Activity of Antibodies

An Assay for Bacterial Extracellular Protein-Mediated Cytotoxicity on PMNs

Cytotoxicity Assay of Staphylococcus aureus Strains on Neutrophils following Phagocytosis

Assessment of Opsonophagocytic Killing Activity against Bacterial Pathogens