Name: opsono-adherence assay to evaluate functional antibodies in vaccine development against bacillus anthracis and other encapsulated pathogens
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Description: Watch this Scientific Journal Video about Opsono-Adherence Assay to Evaluate Functional Antibodies in Vaccine Development Against Bacillus anthracis and Other Encapsulated Pathogens at JoVE.com

J. Chua, D. Chabot and A. Friedlander designed the procedures described in the manuscript. J. Chua and T. Putmon-Taylor performed the experiments. D. Chabot performed data analysis. J. Chua wrote the manuscript.
The authors thank Kyle J. Fitts for excellent technical assistance.
The work was supported by the Defense Threat Reduction Agency grant CBM.VAXBT.03.10.RD.015, plan number 921175.
Opinions, interpretations, conclusions and recommendations are those of the authors and are not necessarily endorsed by the U. S. Army. The content of this publication does not necessarily reflect the views or policies of the Department of Defense, nor does mention of trade names, commercial products, or organizations imply endorsement by the U. S. Government.

In compliance with the Animal Welfare Act, Public Health Service policy, and other federal statutes and regulations pertaining to animals and experiments involving animals, the research described here was conducted under an Institutional Animal Care and Use Committee&#8211;approved protocol. The facility where this research was conducted is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care, International and adheres to principles stated in the Guide for the Care and Use of Laborator...

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Capsule based vaccines have been shown to be efficacious against numerous bacterial pathogens, and many are licensed for use in humans25,26,27. These vaccines work by generating antibodies targeting the capsule and many of these studies use the OPKA to show the opsono-phagocytic functions of the antibodies13,14,16,<sup class=...

Recognition, adherence, internalization, and degradation of a pathogen are integral to phagocytosis1, a salient pathway in the host innate immune response first described by Ilya Metchnikoff in 18832,3. Phagocytic leukocytes, as well as other cells of the immune system, are highly discriminatory in their selection of targets; they are able to distinguish between &#8220;infectious non-self&#8221; and &#8220;non-infectious self&#8221; through pathogen associated molecular patterns by their repertoire of pattern recognition receptors (PRRs)4,5. Host recognition of a pathogen may also occur with the binding of host&#160;opsonins, such as complement and antibodies6. This process, called opsonization, coats the pathogen with these molecules, enhancing internalization upon binding to opsonic receptors (e.g., complement and Fc receptors) on phagocytic cells6. For a pathogen to adhere to a phagocyte, collective binding of multiple receptors with their cognate ligands is necessary. Only then can adherence trigger and sustain signaling cascades inside the host cell to initiate internalization6.
Due to the importance of phagocytosis in the clearance of pathogens and prevention of infection, extracellular pathogens have developed numerous ways to subvert this process to prolong their survival. One strategy of importance is the production of an anionic polymeric (e.g., polysaccharide or polyamino acid) capsule which is anti-phagocytic by virtue of its charge, is poorly immunogenic, and shields molecules on the bacterial envelope from PRRs6,7. Pathogens such as Cryptococcus neoformans and Streptococcus pneumoniae have capsules composed of saccharide polymers, whereas Staphylococcus epidermidis and some Bacillus species produce poly-&#611;-glutamic acid (PGGA)7,8. Yet other pathogens produce capsules that resemble the non-infectious self. For example, Streptococcus pyogenes and a pathogenic strain of B. cereus have a hyaluronic acid capsule that is not only anti-phagocytic but which also may not be recognized as foreign by the immune system9,10.
Conjugation of capsule to carrier proteins converts them from poor, T-independent antigens into highly immunogenic T-dependent antigens that can induce high serum anti-capsule antibody titers11,12. This strategy is employed for licensed vaccines against S. pneumoniae, Haemophilus influenzae, and Neisseria meningitides11. The opsonic activities of anti-capsule antibodies have commonly been evaluated by&#160;opsono-phagocytic killing assays (OPKA)13,14,15,16. These&#160;assays test&#160;whether functional antibodies can trigger phagocytosis and killing14. However, the use of OPKA with infectious pathogens, such as Tier 1 Biological Select Agents and Toxins (BSAT), including B. anthracis17, is hazardous and presents security risks; these assays necessitate extensive handling of a select agent. Select agent handling can only be done in restricted biosafety level 3 (BSL-3) laboratories; work in these areas demands protracted operating procedures due to the numerous safety and security precautions that must be followed. BSL-3 laboratories are also typically not equipped with the specialized equipment used for OPKA work, such as microscopes and cytometers. Thus, we developed an alternative assay based on the use of inactivated bacteria18,19. We refer to this as an opsono-adherence assay (OAA) that is not dependent on internalization and killing as assay outputs; instead, adherence of opsonized inactivated pathogens is used as an index of phagocytosis. Mechanistically, OAA is a suitable substitute because adherence occurs a priori and is intimately intertwined with internalization and intracellular killing. From a biosafety perspective, OAA is preferred because it requires minimal handling of an infectious agent, is experimentally of shorter duration than OPKA, and can be performed in BSL-2 laboratories after a stock of the inactivated pathogen has been produced and transferred.
We demonstrate the utilization of OAA to examine the opsonic function of anti-capsule antibodies found in sera of non-human primates (NHPs) vaccinated with a capsule conjugate [i.e. PGGA from B. anthracis conjugated to the outer membrane protein complex (OMPC) of Neisseria meningitides]20. Serum opsonized bacilli were incubated with an adherent mouse macrophage cell line, RAW 264.7. After fixation, the cell monolayer and adherent bacilli were imaged by fluorescence microscopy. Bacterial adherence increased when the bacilli were incubated with serum from NHPs vaccinated with the capsule conjugate compared to control serum20. Adherence correlated with survival of the anthrax challenge20,21. Thus, the use of OAA characterized the function of anti-capsule antibodies and greatly facilitated testing of our vaccine candidate.&#160;&#160; &#160;

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			<td height="20" style="height:20px;width:380px;">0.20 &#181;m syringe filter (25mm, regenerated cellulose)</td>
			<td style="width:483px;">Corning, Corning, NY</td>
			<td style="width:267px;">431222</td>
			<td style="width:391px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">10 mL syringe (Luer-Lok tip)</td>
			<td>BD, Franklin Lakes, NJ</td>
			<td>302995</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">15&#181; 96 well black plates (plate #1 for imaging)</td>
			<td>In Vitro Scientific, Sunnyvale, CA</td>
			<td>P96-1-N</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">16% paraformaldehyde</td>
			<td>Electron Microscopy Science, Hatfield, PA</td>
			<td>15710</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">75 cm sq. tissue culture treated flask</td>
			<td>Corning, Corning, NY</td>
			<td>430641</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Agar (powder)</td>
			<td>Sigma-Aldrich, St. Louis, MO</td>
			<td>A1296</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Baby Rabbit Complement</td>
			<td>Cedarlane Labs, Burlington, NC</td>
			<td>CL3441</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bacto Yeast Extract</td>
			<td>BD, Sparks, MD</td>
			<td>288620</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BBL Brain Heart Infusion (BHI)</td>
			<td>BD, Sparks, MD</td>
			<td>211059</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Blood Agar (TSA with Sheep Blood) plates</td>
			<td>Remel, Lenexa, KS</td>
			<td>R01198</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell scraper</td>
			<td>Sarstedt, Newton, NC</td>
			<td>83.183</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Costar 96 well cell culture plates (plates #2 &#38; 3 for dilutions)</td>
			<td>Corning, Corning, NY</td>
			<td>3596</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cover glass</td>
			<td>Electron Microscopy Science, Hatfield, PA</td>
			<td>72200-10</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Difco Nutrient Broth</td>
			<td>BD, Sparks, MD</td>
			<td>234000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dulbecco&#39;s Modified Eagle Medium (DMEM), high glucose</td>
			<td>Gibco, Thermo Fisher Scientific, Waltham, MA</td>
			<td>11965-092</td>
			<td>contains 4500 mg/L glucose, 4 mM L-glutamine, Phenol Red</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EVOS FL Auto Cell Imaging System (fluorescence microscope)</td>
			<td>Life Technologies, Thermo Fisher Scientific, Waltham, MA</td>
			<td>AMAFD1000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal Bovine Serum</td>
			<td>Hyclone, GE Healthcare Life Sciences, South Logan, UT</td>
			<td>SH30071.03</td>
			<td>not gamma irradiated, not heat inactivated</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fluorescein isothiocyanate</td>
			<td>Invitrogen, Thermo Fisher Scientific, Waltham, MA</td>
			<td>F143</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HCS Cell Mask Orange Cell Stain</td>
			<td>Invitrogen, Thermo Fisher Scientific, Waltham, MA</td>
			<td>H32713</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">hemocytometer (Improved Neubauer)</td>
			<td>Hausser Scientific, Horsham, PA</td>
			<td>3900</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">India Ink solution</td>
			<td>BD, Sparks, MD</td>
			<td>261194</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">L- glutamine (200 mM)</td>
			<td>Gibco, Thermo Fisher Scientific, Waltham MA</td>
			<td>25030081</td>
			<td>supplement medium with additional 2mM L-glutamine</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Nikon Eclipse TE2000-U (inverted compound microscope)</td>
			<td>Nikon Instruments, Melville, NY</td>
			<td>TE2000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PBS without Calcium or Magnesium</td>
			<td>Lonza, Walkersville, MD</td>
			<td>17-516F</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin-Streptomycin Solution, 100x</td>
			<td>Hyclone, GE Healthcare Life Sciences, South Logan, UT</td>
			<td>SV30010</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">petri dishes (100 x 15 mm)</td>
			<td>Falcon, Corning, Durham, NC</td>
			<td>351029</td>
			<td>for agar plates</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RAW 264.7 macrophage cell line (Tib47)</td>
			<td>ATCC, Manassas, VA</td>
			<td>ATCC TIB-71</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Slides</td>
			<td>VWR, Radnor, PA</td>
			<td>16004-422</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium Bicarbonate</td>
			<td>Sigma-Aldrich, St. Louis, MO</td>
			<td>S5761</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trypan Blue Solution (0.4%)</td>
			<td>Sigma-Aldrich, St. Louis, MO</td>
			<td>T8154</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Zeiss 700 Laser Scanning Microscopy (confocal microscope)</td>
			<td>Carl Zeiss Microimaging, Thornwood, NY</td>
			<td>4109001865956000</td>
			<td></td>
		</tr>
	</tbody>
</table>

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;

This section shows representative micrographs collected during an OAA experiment along with results&#160;showing that the OAA can be used to examine the biological function of antibodies. Here, the assay was successfully used to evaluate the efficacy of an anthrax vaccine candidate. It is critical to verify the state of encapsulation on the bacilli as little to no encapsulation causes them to adhere to host cells, producing a high background. Figure 1 is an image of B. anthracis Ame...

Watch this Scientific Journal Video about Opsono-Adherence Assay to Evaluate Functional Antibodies in Vaccine Development Against Bacillus anthracis and Other Encapsulated Pathogens at JoVE.com

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.

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 ...

The Opsono-Adherence Assay analyzes the presence of functional oposonic antibodies produced in animals after vaccination, which have been shown to correlate with the efficacy of several licensed human vaccines. This assay is preferred for use with biological select agents and other infectious pathogens and can be performed in a BSL-1 or BSL-2 lab after the inactivated BSL-3 agent is transferred. Antibodies against bacterial capsules, including the Bacillus anthracis capsule, have been demonstrated to be efficacious in animal models of infection.Opsono-Adherence assays functionally evaluate antibodies without infecting animals. Follow all institutional safety and security procedures when working with encapsulated virulent strains of Bacillus anthracis. For this video, we're using an inactivated strain under biosafety level two conditions.Begin this procedure with preparation of RAW 264.7 cells, as described in the text protocol. To plate the cells for the opsono-adherence assay, replace the flask with fresh medium and use a cell scraper to gently scrape cells off the flask. Pipette up and down to break up the cell clumps for easier cell counting.Plate 14, 000 cells per well in a 96-well 0.15 micron thick glass flat bottom plate. Allow cells to grow for three days, replacing spent medium one day before performing the OAA. After three days of incubation, the number of cells should be approximately 50, 000 cells per well.To begin the assay, prepare a master plate of Bacillus anthracis from a spore stock by streaking it for isolation on an NBY agar plate. For demonstration purposes, a sheep blood agar plate was used. Incubate at 37 degrees Celsius for one day before culturing in broth.Then, inoculate 30 milliliters of culture broth in a vented 250 milliliter flask with several colonies of the bacilli. Shake the broth culture at 125 RPM, 37 degrees Celsius, with 20%CO2 and humidity for 18 to 24 hours. Use negative staining with India ink to ensure sufficient encapsulation, and that Bacilli are in short chains prior to fixation.To do so, mix five microliters of bacterial suspension with two microliters of India ink on a microscope slide. Then, place a one or 1.5 micron thick cover glass on top of the suspension without creating bubbles. Observe the bacterial suspension using a 40 times to 100 times oil objective lens on a light microscope.A thick capsule excludes India ink. Ensure that the Bacilli are encapsulated by observing a zone of clearance surrounding each bacterium. Also ensure that the Bacilli chains are short.To determine Colony Forming Units, or CFUs, serially dilute 100 microliters of culture one to 10 in PBS solution, and plate culture dilutions on sheep blood agar plates. Incubate for 12 to 18 hours at 37 degrees Celsius. Count CFUs, and determine the number of bacteria per milliliter of culture.After ensuring that the Bacilli are sufficiently encapsulated, diluted, and plated, fix the remaining bacterial culture by adding 10 milliliters of 16%paraformaldehyde to the 30 milliliters of culture at a final concentration of 4%paraformaldehyde. Transfer the volume to a 50 milliliter conical tube. Then, slowly agitate on a shaker at ambient temperature for seven days.To test for sterility, transfer four milliliters, or 10%volume, of fixed bacterial suspension to a 50 milliliter conical tube. Wash the suspension by adding PBS, and pelleting at a minimum of 3000 G for 45 minutes. Then remove the supernatant, ensuring that the fluffy pellet is not disturbed.After repeating this wash at least two times, resuspend the fixed sample in four milliliters PBS. It's necessary to remove all fixative during the washes so that the fixative does not interfere with the viability testing. If necessary, increase the number of washes.For viability testing, inoculate 40 milliliters of a bacterial growth medium with four milliliters of washed suspension and incubate at 37 degrees Celsius for seven days. Check optical density at 600 nanometers in a spectrophotometer before and after seven days to measure turbidity. After seven days of incubation in broth culture, plate at least 100 microliters of broth onto an agar plate and incubate for another seven days.If no increase in turbidity and no growth on plates are seen, the original culture is considered sterile, and maybe transferred to a BSL-2 laboratory. Follow institutional guidelines for transfer approval. Wash the inactivated bacterial stock three times in PBS to remove fixative.Add FITC solution at a final concentration of 75 micrograms per milliliter. Slowly agitate the mixture for 18 to 24 hours at four degrees Celsius. Wash the bacteria as before to remove excess FITC.After the final wash, resuspend the Bacilli in PBS in the same start volume. and store Bacilli in minus 20 degrees Celsius until use. Heat-inactivate a small volume of test sera from non-human primates at 56 degrees Celsius for 30 minutes in a heat block or PCR machine.Fall and wash FITC-labeled bacteria with PBS two times by pelleting at 15, 000 times G for three to five minutes. Resuspend in PBS in the same start volume. In a 96-well round-bottom plate, serially dilute the test sera in cell medium.In another 96-well round-bottom plate, add 10 microliters of diluted test sera into each well. Then, add 10 microliters of freshly reconstituted baby rabbit compliment. To plate number three, add the appropriate volume of FITC-labeled Bacilli for a multiplicity of infection of 20.For example, at a volume containing 50, 000 times 20 Bacilli per 50, 000 cells. Top off each well in plate number three with the appropriate volume of cell medium to a total of 105 microliters. Incubate plate number three at 37 degrees Celsius for 30 minutes in the presence of 5%CO2 with humidity to opsonize Bacilli.Place plate number one, which contains adherence cells, on a 37 degrees Celsius heat block. Use a multi-channel pipetter to remove spent medium from wells, and quickly replace with 100 microliters of opsonize bacteria from plate number three, ensuring no bubbles are generated in the wells during pipetting. To not disturb the cell monolayer, add volume to the sides of the wells.Incubate plate number one at 37 degrees Celsius with 5%CO2 and humidity for 30 minutes for adherence. Wash each well five times with 150 microliters of PBS, on top of the 37 degrees Celsius heat block, to remove unattached Bacilli. Pipette to the side of the wells to ensure that the cells are not accidentally dispersed during washing.Add 150 microliters of 4%paraformaldehyde to each well, and fix the cells for one hour. After washing the cells twice with PBS, mix two microliters of high-content screening orange cells stain in 10 milliliters of PBS. Then, add 150 microliters of this staining solution to fix cells, and incubate for 30 minutes at ambient temperature.Place plate number one on the stage and focus on the cells using brightfield or phase contrast. In the Acquisition tab, select 96-well format as the plate setting. Then, select Channel settings.Next, select setting to Tile mode. Indicate the number of images to be acquired. Change the Acquisition mode to black and white or monochrome, and set the Binning to at least two by two.Turn on the Autofocus or focusing module. Indicate how often the autofocusing function is to be performed. Next, turn on the Z-Stack function, if available.Indicate the number of Z-Stacks that will capture the thickness of the cell monolayer and adherent Bacilli. Designate a file folder to save the images to. Now, run the automated imaging program.Perform data collection and analysis as described in the text protocol. The Ames strain of bacillus anthracis was negatively stained with India ink to examine encapsulation. The state of encapsulation must be verified, as little to no encapsulation causes them to adhere to host cells without antibodies, producing a high background.Fluorescence intensity and photo bleaching characteristics of the Bacilli was examined using Confocal Microscopy. It is critical to verify that the fluorescence of the Bacilli do not significantly fade over time, because the Bacilli will undergo long exposures to light during imaging. The maximum projection images of Bacillus anthracis attach to the cell monolayers are shown here.These are representative fields of view that were counted and scored for the opsono-adherence assay. The increase in attachment of Bacilli incubated with capsule conjugate antiserum is due to the presence of anti-capsule antibodies that opsonized the Bacilli. The serum titers of non-human primates vaccinated with 10 and 50 micrograms of capsule conjugate are significantly higher than the titers for conjugate and capsule alone.We are developing a similar assay that uses flow cytometry and human neutrophils, which are nonadherent cells, to measure opsonic activity and also to quantitate capsule on the surface of bacteria. After watching this video, you should have a good understanding of the opsono-adherence assay. Don't forget that working with BSL-3 pathogens is extremely hazardous.Institutional guidelines and safety precautions must be strictly followed.

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