Opsono-Adherence Assay to Evaluate Functional Antibodies in Vaccine Development Against Bacillus anthracis and Other Encapsulated Pathogens
Summary
The opsono-adherence assay is an alternative method to the opsono-phagocytic killing assay to evaluate the opsonic functions of antibodies in vaccine development.
Abstract
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 opsono-phagocytic killing assays. An advantage of the opsono-adherence 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.
Introduction
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 “infectious non-self” and “non-infectious self” 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 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-ɣ-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 opsono-phagocytic killing assays (OPKA)13,14,15,16. These assays test 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.
Protocol
Representative Results
Discussion
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=…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| 0.20 µm syringe filter (25mm, regenerated cellulose) | Corning, Corning, NY | 431222 | |
| 10 mL syringe (Luer-Lok tip) | BD, Franklin Lakes, NJ | 302995 | |
| 15µ 96 well black plates (plate #1 for imaging) | In Vitro Scientific, Sunnyvale, CA | P96-1-N | |
| 16% paraformaldehyde | Electron Microscopy Science, Hatfield, PA | 15710 | |
| 75 cm sq. tissue culture treated flask | Corning, Corning, NY | 430641 | |
| Agar (powder) | Sigma-Aldrich, St. Louis, MO | A1296 | |
| Baby Rabbit Complement | Cedarlane Labs, Burlington, NC | CL3441 | |
| Bacto Yeast Extract | BD, Sparks, MD | 288620 | |
| BBL Brain Heart Infusion (BHI) | BD, Sparks, MD | 211059 | |
| Blood Agar (TSA with Sheep Blood) plates | Remel, Lenexa, KS | R01198 | |
| Cell scraper | Sarstedt, Newton, NC | 83.183 | |
| Costar 96 well cell culture plates (plates #2 & 3 for dilutions) | Corning, Corning, NY | 3596 | |
| Cover glass | Electron Microscopy Science, Hatfield, PA | 72200-10 | |
| Difco Nutrient Broth | BD, Sparks, MD | 234000 | |
| Dulbecco's Modified Eagle Medium (DMEM), high glucose | Gibco, Thermo Fisher Scientific, Waltham, MA | 11965-092 | contains 4500 mg/L glucose, 4 mM L-glutamine, Phenol Red |
| EVOS FL Auto Cell Imaging System (fluorescence microscope) | Life Technologies, Thermo Fisher Scientific, Waltham, MA | AMAFD1000 | |
| Fetal Bovine Serum | Hyclone, GE Healthcare Life Sciences, South Logan, UT | SH30071.03 | not gamma irradiated, not heat inactivated |
| Fluorescein isothiocyanate | Invitrogen, Thermo Fisher Scientific, Waltham, MA | F143 | |
| HCS Cell Mask Orange Cell Stain | Invitrogen, Thermo Fisher Scientific, Waltham, MA | H32713 | |
| hemocytometer (Improved Neubauer) | Hausser Scientific, Horsham, PA | 3900 | |
| India Ink solution | BD, Sparks, MD | 261194 | |
| L- glutamine (200 mM) | Gibco, Thermo Fisher Scientific, Waltham MA | 25030081 | supplement medium with additional 2mM L-glutamine |
| Nikon Eclipse TE2000-U (inverted compound microscope) | Nikon Instruments, Melville, NY | TE2000 | |
| PBS without Calcium or Magnesium | Lonza, Walkersville, MD | 17-516F | |
| Penicillin-Streptomycin Solution, 100x | Hyclone, GE Healthcare Life Sciences, South Logan, UT | SV30010 | |
| petri dishes (100 x 15 mm) | Falcon, Corning, Durham, NC | 351029 | for agar plates |
| RAW 264.7 macrophage cell line (Tib47) | ATCC, Manassas, VA | ATCC TIB-71 | |
| Slides | VWR, Radnor, PA | 16004-422 | |
| Sodium Bicarbonate | Sigma-Aldrich, St. Louis, MO | S5761 | |
| Trypan Blue Solution (0.4%) | Sigma-Aldrich, St. Louis, MO | T8154 | |
| Zeiss 700 Laser Scanning Microscopy (confocal microscope) | Carl Zeiss Microimaging, Thornwood, NY | 4109001865956000 |
Referenzen
- Walters, M. N., Papadimitriou, J. M. Phagocytosis: a review. CRC Critical Reviews in Toxicology. 5 (4), 377-421 (1978).
- Metschnikoff, E. Untersuchurgen uber die intracellulare Verdauung, bei wirbellosen Thieren. Arbeiten aus dem Zoologischen Instituten der Universität Wien. 5, 144 (1883).
- Tauber, A. I. Metchnikoff and the phagocytosis theory. Nature Reviews Molecular Cell Biology. 4 (11), 897-901 (2003).
- Janeway, C. A. The immune system evolved to discriminate infectious nonself from noninfectious self. Immunology Today. 13 (1), 11-16 (1992).
- Kumagai, Y., Akira, S. Identification and functions of pattern-recognition receptors. Journal of Allergy and Clinical Immunology. 125 (5), 985-992 (2010).
- Flannagan, R. S., Jaumouille, V., Grinstein, S. The cell biology of phagocytosis. Annual Review of Pathology. 7, 61-98 (2012).
- Candela, T., Fouet, A. Poly-gamma-glutamate in bacteria. Molecular Microbiology. 60 (5), 1091-1098 (2006).
- Kocianova, S., et al. Key role of poly-gamma-DL-glutamic acid in immune evasion and virulence of Staphylococcus epidermidis. Journal of Clinical Investigation. 115 (3), 688-694 (2005).
- Rothbard, S. Protective effect of hyaluronidase and type-specific anti-M serum on experimental group A Streptococcus infection in mice. Journal of Experimental Medicine. 88 (3), 325-342 (1948).
- Oh, S. Y., Budzik, J. M., Garufi, G., Schneewind, O. Two capsular polysaccharides enable Bacillus cereus G9241 to cause anthrax-like disease. Molecular Microbiology. 80 (2), 455-470 (2011).
- Knuf, M., Kowalzik, F., Kieninger, D. Comparative effects of carrier proteins on vaccine-induced immune response. Vaccine. 29 (31), 4881-4890 (2011).
- Wang, T. T., Lucas, A. H. The capsule of Bacillus anthracis behaves as a thymus-independent type 2 antigen. Infection & Immunity. 72 (9), 5460-5463 (2004).
- Vogel, L., et al. Quantitative flow cytometric analysis of opsonophagocytosis and killing of nonencapsulated Haemophilus influenzae by human polymorphonuclear leukocytes. Clinical and Diagnostic Laboratory Immunology. 1 (4), 394-400 (1994).
- Salehi, S., Hohn, C. M., Penfound, T. A., Dale, J. B. Development of an Opsonophagocytic Killing Assay Using HL-60 Cells for Detection of Functional Antibodies against Streptococcus pyogenes. mSphere. 3 (6), 00617-00618 (2018).
- Wang, Y., et al. Novel Immunoprotective Proteins of Streptococcus pneumoniae Identified by Opsonophagocytosis Killing Screen. Infection & Immunity. 86 (9), (2018).
- Humphries, H. E., et al. Seroprevalence of Antibody-Mediated, Complement-Dependent Opsonophagocytic Activity against Neisseria meningitidis Serogroup B in England. Clinical and Vaccine Immunology. 22 (5), 503-509 (2015).
- Jansen, W. T., et al. Use of highly encapsulated Streptococcus pneumoniae strains in a flow-cytometric assay for assessment of the phagocytic capacity of serotype-specific antibodies. Clinical and Diagnostic Laboratory Immunology. 5 (5), 703-710 (1998).
- Wang, T. T., Fellows, P. F., Leighton, T. J., Lucas, A. H. Induction of opsonic antibodies to the gamma-D-glutamic acid capsule of Bacillus anthracis by immunization with a synthetic peptide-carrier protein conjugate. FEMS Immunology and Medical Microbiology. 40 (3), 231-237 (2004).
- Chabot, D. J., et al. Protection of rhesus macaques against inhalational anthrax with a Bacillus anthracis capsule conjugate vaccine. Vaccine. 34 (34), 4012-4016 (2016).
- Chabot, D. J., et al. Efficacy of a capsule conjugate vaccine against inhalational anthrax in rabbits and monkeys. Vaccine. 30 (5), 846-852 (2012).
- Chua, J., et al. Formaldehyde and Glutaraldehyde Inactivation of Bacterial Tier 1 Select Agents in Tissues. Emerging Infectious Diseases. 25 (5), 919-926 (2019).
- . Guide for the Care and Use of Laboratory Animals (Eighth Edition) Available from: https://grants.nih.gov/grants/olaw/guide-for-the-care-and-use-of-laboratory-animals.pdf (2011)
- Durando, P., et al. Experience with pneumococcal polysaccharide conjugate vaccine (conjugated to CRM197 carrier protein) in children and adults. Clinical Microbiology and Infection. 19, 1-9 (2013).
- Adams, W. G., et al. Decline of childhood Haemophilus influenzae type b (Hib) disease in the Hib vaccine era. JAMA. 269 (2), 221-226 (1993).
- Balmer, P., Borrow, R., Miller, E. Impact of meningococcal C conjugate vaccine in the UK. Journal of Medical Microbiology. 51 (9), 717-722 (2002).
- Chen, M., et al. Induction of opsonophagocytic killing activity with pneumococcal conjugate vaccine in human immunodeficiency virus-infected Ugandan adults. Vaccine. 26 (38), 4962-4968 (2008).
- Paschall, A. V., Middleton, D. R., Avci, F. Y. Opsonophagocytic Killing Assay to Assess Immunological Responses Against Bacterial Pathogens. Journal of Visualized Experiments. (146), e59400 (2019).
- Ezzell, J. W., Welkos, S. L. The capsule of Bacillus anthracis, a review. Journal of Applied Microbiology. 87 (2), 250 (1999).
- Hanna, P. C., Acosta, D., Collier, R. J. On the role of macrophages in anthrax. Proceedings of the National Academies of Sciences of the United States of America. 90 (21), 10198-10201 (1993).
- Fleck, R. A., Romero-Steiner, S., Nahm, M. H. Use of HL-60 cell line to measure opsonic capacity of pneumococcal antibodies. Clinical and Diagnostic Laboratory Immunology. 12 (1), 19-27 (2005).
- Chua, J., Deretic, V. Mycobacterium tuberculosis reprograms waves of phosphatidylinositol 3-phosphate on phagosomal organelles. Journal of Biological Chemistry. 279 (35), 36982-36992 (2004).
- Chua, J., et al. pH Alkalinization by Chloroquine Suppresses Pathogenic Burkholderia Type 6 Secretion System 1 and Multinucleated Giant Cells. Infection & Immunity. 85 (1), (2017).
- Ober, R. J., Radu, C. G., Ghetie, V., Ward, E. S. Differences in promiscuity for antibody-FcRn interactions across species: implications for therapeutic antibodies. International Immunology. 13 (12), 1551-1559 (2001).
- Sorman, A., Zhang, L., Ding, Z., Heyman, B. How antibodies use complement to regulate antibody responses. Molecular Immunology. 61 (2), 79-88 (2014).
- Henckaerts, I., Durant, N., De Grave, D., Schuerman, L., Poolman, J. Validation of a routine opsonophagocytosis assay to predict invasive pneumococcal disease efficacy of conjugate vaccine in children. Vaccine. 25 (13), 2518-2527 (2007).
- Wolf, J. J. . Special Considerations for the Nonclinical Safety Assessment of Vaccines. , 243-255 (2013).
