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1
Lin, J. S., M. K. Park, and M. H. Nahm. "Chromogenic Assay Measuring Opsonophagocytic Killing Capacities of Antipneumococcal Antisera." Clinical Diagnostic Laboratory Immunology 8, no. 3 (May 1, 2001): 528–33. http://dx.doi.org/10.1128/cdli.8.3.528-533.2001.
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ABSTRACT Assays measuring opsonophagocytic killing capacity of immune sera are good surrogate assays for assessing pneumococcal vaccine responses, but they are tedious to perform primarily because the enumeration of surviving bacteria requires the counting of individual bacterial colonies. To overcome this limitation, we have developed a simple and rapid chromogenic assay for estimating the number of surviving bacteria. In this method, the conventional opsonophagocytic killing assays were performed in microtiter wells with differentiated HL-60 cells as phagocytes. At the end of the assay the reaction mixture was cultured for an additional 4.5 h to increase the number of bacteria. After the short culture, XTT (3,3′-[1{(phenylamino)carbonyl}-3,4-tetrazolium]-bis[4-methoxy-6-nitro] benzene sulfonic acid hydrate) and coenzyme Q were added to the wells and the optical density at 450 nm was measured. Our study shows that changes in the optical density were proportional to the number of CFU of live bacteria in the wells. Also, the number of bacteria at the end of the 4.5-h culture was found to be proportional to the original number of bacteria in the wells. When the performance of the chromogenic assay was evaluated by measuring the opsonizing titers ofStreptococcus pneumoniae serotypes 6B and 19F, the sensitivity and precision of the new method were similar to those of the conventional opsonization assay employing the colony counting method. Furthermore, the results of this chromogenic assay obtained with 33 human sera correlate well with those obtained with the conventional colony counting method (R > 0.90) for the two serotypes (6B and 19F). Thus, this simple chromogenic assay would be useful in rapidly measuring the capacities of antisera to opsonize pneumococci.
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Nahm, Moon H., David E. Briles, and Xinhong Yu. "Development of a multi-specificity opsonophagocytic killing assay." Vaccine 18, no. 24 (June 2000): 2768–71. http://dx.doi.org/10.1016/s0264-410x(00)00044-x.
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Nolan, Katrina M., Marie E. Bonhomme, Christina J. Schier, Tina Green, Joseph M. Antonello, and Rocio D. Murphy. "Optimization and validation of a microcolony multiplexed opsonophagocytic killing assay for 15 pneumococcal serotypes." Bioanalysis 12, no. 14 (July 2020): 1003–20. http://dx.doi.org/10.4155/bio-2020-0024.
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Background: To streamline and improve throughput, the agar-based multiplexed opsonophagocytic killing assay (MOPA) was optimized and validated on a microcolony platform for use in the Phase III clinical trial program for V114, an MSD 15-valent pneumococcal conjugate vaccine candidate. Results & methodology: The precision, dilutional linearity and specificity of the microcolony MOPA (mMOPA) were assessed for each serotype in validation experiments. All prespecified acceptance criteria on assay performance were satisfied. Accuracy was assessed by testing 007sp and the US FDA reference panel and comparing to consensus values. The mMOPA produced comparable results to other opsonophagocytic killing assays/MOPAs. Conclusion: The mMOPA is suitable for measuring functional antibodies in adult and pediatric samples. Benefits include throughput, reduced analyst-to-analyst variability and automation potential.
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Hu, Branda T., Xinhong Yu, Thomas R. Jones, Carol Kirch, Sarah Harris, Stephen W. Hildreth, Dace V. Madore, and Sally A. Quataert. "Approach to Validating an Opsonophagocytic Assay for Streptococcus pneumoniae." Clinical Diagnostic Laboratory Immunology 12, no. 2 (February 2005): 287–95. http://dx.doi.org/10.1128/cdli.12.2.287-295.2005.
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ABSTRACT Streptococcus pneumoniae (pneumococcus) polysaccharide serotype-specific antibodies that have opsonophagocytic activity are considered a primary mechanism of host defense against pneumococcal disease. In vitro opsonophagocytic assays (OPAs) with antibody and complement to mediate opsonophagocytic killing of bacteria have been designed and developed as an adjunct to the standardized serum immunoglobulin G antipneumococcal capsular polysaccharide enzyme immunoassay to assess the effectiveness of pneumococcal vaccines. OPA presents challenges for assay standardization and assay precision due to the multiple biologically active and labile components involved in the assay, including human polymorphonuclear leukocytes or cultured effector cells, bacteria, and complement. Control of these biologically labile components is critical for consistent assay performance. An approach to validating the performance of the assay in accordance with International Conference for Harmonization guidelines, including its specificity, intermediate precision, accuracy, linearity, and robustness, is presented. Furthermore, we established parameters for universal reagents and standardization of the use of these reagents to ensure the interlaboratory reproducibility and validation of new methodologies.
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Winter, Linda E., and Stephen J. Barenkamp. "Antibodies Specific for the High-Molecular-Weight Adhesion Proteins of Nontypeable Haemophilus influenzae Are Opsonophagocytic for both Homologous and Heterologous Strains." Clinical and Vaccine Immunology 13, no. 12 (October 4, 2006): 1333–42. http://dx.doi.org/10.1128/cvi.00221-06.
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ABSTRACT The HMW1/HMW2-like adhesion proteins of nontypeable Haemophilus influenzae (NTHI) are expressed by 75% of NTHI strains. Antibodies directed against these proteins are opsonophagocytic in vitro and are protective in an animal model of infection. The objective of the present study was to determine the opsonophagocytic activity of high-titer anti-HMW1/HMW2 immune sera against both homologous and heterologous NTHI strains. Chinchillas were immunized with purified HMW1/HMW2-like proteins from five prototype NTHI strains. Serum opsonophagocytic activity was monitored in an assay that uses a human promyelocytic cell line, HL-60, as the source of phagocytic cells. Preimmune sera did not demonstrate opsonophagocytic killing of any strains. In contrast, the immune sera demonstrated killing of the five homologous NTHI strains at titers ranging from 1:320 to 1:640. The immune sera also demonstrated killing of eight heterologous NTHI strains that express HMW1/HMW2-like proteins at titers ranging from 0 to 1:640. Killing of heterologous strains sometimes demonstrated a prozone phenomenon. None of the immune sera killed NTHI strains that did not express HMW1/HMW2-like proteins. Adsorption of immune sera with HMW1/HMW2-like proteins purified from either homologous or heterologous NTHI strains eliminated opsonophagocytic killing of homologous strains in most cases. These data demonstrate that antibodies produced following immunization with the HMW1/HMW2-like proteins are opsonophagocytic for both homologous and heterologous NTHI and strongly suggest that common epitopes recognized by functionally active antibodies exist on the HMW1/HMW2-like proteins of unrelated NTHI strains. The results argue for the continued investigation of the HMW1/HMW2-like proteins as potential vaccine candidates for the prevention of NTHI disease.
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Ramachandran, Girish, Mary Adetinuke Boyd, Jennifer MacSwords, Ellen E. Higginson, Raphael Simon, James E. Galen, Marcela F. Pasetti, Myron M. Levine, and Sharon M. Tennant. "Opsonophagocytic Assay To Evaluate Immunogenicity of Nontyphoidal Salmonella Vaccines." Clinical and Vaccine Immunology 23, no. 6 (March 30, 2016): 520–23. http://dx.doi.org/10.1128/cvi.00106-16.
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ABSTRACTNontyphoidalSalmonella(NTS) invasive infections are an important cause of morbidity and mortality in sub-Saharan Africa. Several vaccines are in development to prevent these infections. We describe an NTS opsonophagocytic killing assay that uses HL-60 cells and baby rabbit complement to quantify functional antibodies elicited by candidate NTS vaccines.
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Jones, Scott, Nicole J. Moreland, Marta Zancolli, Jeremy Raynes, Jacelyn M. S. Loh, Pierre R. Smeesters, Shiranee Sriskandan, Jonathan R. Carapetis, John D. Fraser, and David Goldblatt. "Development of an opsonophagocytic killing assay for group a streptococcus." Vaccine 36, no. 26 (June 2018): 3756–63. http://dx.doi.org/10.1016/j.vaccine.2018.05.056.
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Choi, Min Joo, Ji Yun Noh, Hee Jin Cheong, Woo Joo Kim, Shun-Mei Lin, Yong Zhi, Jae Hyang Lim, Sangyong Lim, Ho Seong Seo, and Joon Young Song. "Development of a multiplexed opsonophagocytic killing assay (MOPA) for group BStreptococcus." Human Vaccines & Immunotherapeutics 14, no. 1 (October 30, 2017): 67–73. http://dx.doi.org/10.1080/21645515.2017.1377379.
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Wang, D., and S. J. Soong. "Comparisons of titer estimation methods for multiplexed pneumococcal opsonophagocytic killing assay." Computational Statistics & Data Analysis 52, no. 11 (July 2008): 5022–32. http://dx.doi.org/10.1016/j.csda.2008.04.025.
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Kim, Jean O., Sandra Romero-Steiner, Uffe B. Skov Sørensen, Jens Blom, M. Carvalho, S. Barnard, George Carlone, and Jeffrey N. Weiser. "Relationship between Cell Surface Carbohydrates and Intrastrain Variation on Opsonophagocytosis ofStreptococcus pneumoniae." Infection and Immunity 67, no. 5 (May 1, 1999): 2327–33. http://dx.doi.org/10.1128/iai.67.5.2327-2333.1999.
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ABSTRACT Streptococcus pneumoniae undergoes spontaneous phase variation between a transparent and an opaque colony phenotype, the latter being more virulent in a murine model of sepsis. Opaque pneumococci have previously been shown to express lower amounts of C polysaccharide (cell wall teichoic acid) and in this study were shown to have a higher content of capsular polysaccharide by immunoelectron microscopy. This report then examined the relationship between expression of these two cell surface carbohydrate structures and their relative contribution to the increased virulence of opaque variants. Comparison of genetically related strains showed that the differential content of capsular polysaccharide did not affect the amount of teichoic acid as measured by a capture enzyme-linked immunosorbent assay (ELISA). In contrast, when the teichoic acid structure was altered by replacing choline in the growth medium with structural analogs, the quantity of capsular polysaccharide as measured by a capture ELISA was decreased, demonstrating a linkage in the expression of the two surface carbohydrate structures. A standardized assay was used to assess the relative contribution of cell surface carbohydrates to opsonophagocytosis. The opaque variants required 1.2- to 30-fold more immune human serum to achieve 50% opsonophagocytic killing than did related transparent variants (types 6B and 9V). The opsonophagocytic titer was proportional to the quantity of capsular polysaccharide rather than teichoic acid. The major factor in binding of the opsonin, C-reactive protein (CRP), was also the amount of capsular polysaccharide rather than the teichoic acid ligand. Only for the transparent variant (type 6B), which bound more CRP, was there enhanced opsonophagocytic killing in the presence of this serum protein. Increased expression of capsular polysaccharide, therefore, appeared to be the major factor in the decreased opsonophagocytic killing of opaque pneumococci.
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Winter, Linda E., and Stephen J. Barenkamp. "Antibodies to the HMW1/HMW2 and Hia Adhesins of Nontypeable Haemophilus influenzae Mediate Broad-Based Opsonophagocytic Killing of Homologous and Heterologous Strains." Clinical and Vaccine Immunology 21, no. 5 (February 26, 2014): 613–21. http://dx.doi.org/10.1128/cvi.00772-13.
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ABSTRACTThe HMW1/HMW2 and Hia proteins are highly immunogenic surface adhesins of nontypeableHaemophilus influenzae(NTHi). Approximately 75% of NTHi strains express HMW1/HMW2 adhesins, and most of the remaining 25% express an Hia adhesin. Our objective in this study was to assess the ability of antisera raised against purified HMW1/HMW2 proteins or recombinant Hia proteins to mediate opsonophagocytic killing of a large panel of unrelated NTHi strains. Native HMW1/HMW2 proteins were purified from three HMW1/HMW2-expressing NTHi strains. Recombinant fusion proteins expressing surface-exposed segments of either of two prototype Hia proteins were purified fromEscherichia colitransformants. Immune sera raised in guinea pigs were assessed for their ability to mediate killing of NTHi in an opsonophagocytic assay with the HL-60 phagocytic cell line. The three HMW1/HMW2 antisera mediated killing of 22 of 65, 43 of 65, and 28 of 65 unrelated HMW1/HMW2-expressing NTHi strains, respectively. As a group, the three sera mediated killing of 48 of 65 HMW1/HMW2-expressing strains. The two Hia immune sera mediated killing of 12 of 24 and 13 of 24 unrelated Hia-expressing NTHi strains, respectively. Together, they mediated killing of 15 of 24 Hia-expressing strains. Neither the HMW1/HMW2 nor the Hia antisera mediated killing of NTHi expressing the alternative adhesin type. Antibodies directed against native HMW1/HMW2 proteins and recombinant Hia proteins are capable of mediating broad-based opsonophagocytic killing of homologous and heterologous NTHi strains. A vaccine formulated with a limited number of HMW1/HMW2 and Hia proteins might provide protection against disease caused by most NTHi strains.
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Kozel, Thomas R., Randall S. MacGill, Ann Percival, and Qing Zhou. "Biological Activities of Naturally Occurring Antibodies Reactive with Candida albicans Mannan." Infection and Immunity 72, no. 1 (January 2004): 209–18. http://dx.doi.org/10.1128/iai.72.1.209-218.2004.
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ABSTRACT Sera from normal adult humans may contain high levels of antibody reactive with Candida albicans mannan. This study examined selected biological activities of such antibodies, focusing on sera that were collected from 34 donors and analyzed individually. The results showed that antimannan titers were normally distributed. Reactivity as determined by enzyme-linked immunosorbent assay with serotype A mannan generally paralleled reactivity with serotype B. Analysis of the kinetics for activation of the complement system and deposition of complement component 3 (C3) onto serotype A and serotype B cells showed a decrease in the lag time that occurred before the onset of rapid accumulation of C3 that correlated with increasing antimannan titers. In contrast, there was a decrease in the overall rate of accumulation of C3 on serotype A cells that was strongly correlated with increasing antibody titers; serotype B cells showed no such decrease. An evaluation of the contribution of mannan antibody to opsonophagocytic killing showed that mannan antibody in individual sera and antimannan immunoglobulin G (IgG) affinity purified from human plasma contributed to killing by neutrophils in a dose-dependent fashion in the absence of a functional complement system. However, affinity-purified antibody in very high concentrations was inhibitory to both complement-dependent and complement-independent opsonophagocytosis, and this finding suggests a prozone-like effect. In contrast, if the complement system was functional, antimannan IgG was not needed for opsonophagocytic killing. These results suggest that naturally occurring mannan antibodies and the complement system are functionally redundant for opsonophagocytic killing by neutrophils.
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Kim, Kyung Hyo, Jigui Yu, and Moon H. Nahm. "Efficiency of a Pneumococcal Opsonophagocytic Killing Assay Improved by Multiplexing and by Coloring Colonies." Clinical Diagnostic Laboratory Immunology 10, no. 4 (July 2003): 616–21. http://dx.doi.org/10.1128/cdli.10.4.616-621.2003.
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ABSTRACT For evaluating pneumococcal vaccines, the opsonophagocytic killing assay (OPKA) is useful as a supplement to the pneumococcal antibody enzyme-linked immunosorbent assay (ELISA). However, evaluations of pneumococcal vaccines require the determination of antibody responses to 7 to 11 serotypes, and the OPKA is tedious to perform and requires more serum than the ELISA. Consequently, the OPKA is infrequently used for evaluating pneumococcal vaccines. To overcome these limitations, we have developed a simple multiplexed (double-serotype) OPKA by using antibiotic-resistant pneumococci for nine serotypes. Serotype 6B, 9V, 19A, and 23F strains were made streptomycin resistant, and serotype 4, 6A, 14, 18C, and 19F strains were made optochin resistant. The multiplexed OPKA was the same as the single-serotype OPKA except for two changes. First, the target bacteria were a mixture of one streptomycin-resistant strain and one optochin-resistant strain. Second, the surviving bacteria of each serotype were enumerated by plating on Todd-Hewitt agar plates with yeast extract and an agar overlay containing the appropriate antibiotics and 2,3,5-triphenyl tetrazolium chloride. The performance of the multiplexed OPKA was evaluated by analyzing 28 serum samples from adults immunized with a 23-valent polysaccharide vaccine by using the single-serotype OPKA and the multiplexed OPKA. The multiplexed OPKA was specific for the desired serotypes. The multiplexed and conventional OPKAs had comparable assay sensitivities and produced results that were highly correlated (r2 values ranging from 0.92 to 0.98) for all nine serotypes. A simple modification of the conventional OPKA produces a multiplexed assay that greatly reduces effort, reagents, and the necessary amount of serum.
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Rose, Charles E., Sandra Romero-Steiner, Robert L. Burton, George M. Carlone, David Goldblatt, Moon H. Nahm, Lindsey Ashton, et al. "Multilaboratory Comparison ofStreptococcus pneumoniaeOpsonophagocytic Killing Assays and Their Level of Agreement for the Determination of Functional Antibody Activity in Human Reference Sera." Clinical and Vaccine Immunology 18, no. 1 (November 17, 2010): 135–42. http://dx.doi.org/10.1128/cvi.00370-10.
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ABSTRACTAntibody-mediated killing ofStreptococcus pneumoniae(pneumococcus) by phagocytes is an important mechanism of protection of the human host against pneumococcal infections. Measurement of opsonophagocytic antibodies by use of a standardized opsonophagocytic assay (OPA) is important for the evaluation of candidate vaccines and required for the licensure of new pneumococcal conjugate vaccine formulations. We assessed agreement among six laboratories that used their own optimized OPAs on a panel of 16 human reference sera for 13 pneumococcal serotypes. Consensus titers, estimated using an analysis-of-variance (ANOVA) mixed-effects model, provided a common reference for assessing agreement among these laboratories. Agreement was evaluated in terms of assay accuracy, reproducibility, repeatability, precision, and bias. We also reviewed four acceptance criterion intervals for assessing the comparability of protocols when assaying the same reference sera. The precision, accuracy, and concordance results among laboratories and the consensus titers revealed acceptable agreement. The results of this study indicate that the bioassays evaluated in this study are robust, and the resultant OPA values are reproducible for the determination of functional antibody titers specific to 13 pneumococcal serotypes when performed by laboratories using highly standardized but not identical assays. The statistical methodologies employed in this study may serve as a template for evaluating future multilaboratory studies.
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Wang, Deli, Robert L. Burton, Moon H. Nahm, and Seng-Jaw Soong. "A Four-Parameter Logistic Model for Estimating Titers of Functional Multiplexed Pneumococcal Opsonophagocytic Killing Assay." Journal of Biopharmaceutical Statistics 18, no. 2 (March 7, 2008): 307–25. http://dx.doi.org/10.1080/10543400701697182.
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Kim, Kyung-Hyo, Ju Young Seoh, and Su Jin Cho. "Phenotypic and Functional Analysis of HL-60 Cells Used in Opsonophagocytic-Killing Assay forStreptococcus pneumoniae." Journal of Korean Medical Science 30, no. 2 (2015): 145. http://dx.doi.org/10.3346/jkms.2015.30.2.145.
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Moraschini, Luca, Irene Passalacqua, Monica Fabbrini, Immaculada Margarit Y Ros, and Fabio Rigat. "Threshold-free estimation of functional antibody titers of a group B streptococcus opsonophagocytic killing assay." Pharmaceutical Statistics 14, no. 3 (February 16, 2015): 189–97. http://dx.doi.org/10.1002/pst.1673.
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Melin, Merit, Hanna Jarva, Lotta Siira, Seppo Meri, Helena Käyhty, and Merja Väkeväinen. "Streptococcus pneumoniae Capsular Serotype 19F Is More Resistant to C3 Deposition and Less Sensitive to Opsonophagocytosis than Serotype 6B." Infection and Immunity 77, no. 2 (December 1, 2008): 676–84. http://dx.doi.org/10.1128/iai.01186-08.
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ABSTRACT The polysaccharide capsule is a major virulence mechanism of Streptococcus pneumoniae, shielding the bacterium from phagocytes. Capsule types may differ in their abilities to resist immune defense. Antibody-mediated complement activation and opsonophagocytosis are crucial in protection against pneumococcus. Conjugate vaccine trials suggest imperfect protection against 19F. We have previously shown that significantly more anti-19F than anti-6B antibody is needed for killing in the opsonophagocytic assay (OPA). In this study, we explored whether the amount of C3 deposited on serotype 6B and 19F pneumococcal strains reflects their sensitivity to opsonophagocytosis. We compared clinical 6B and 19F nasopharyngeal, middle ear, and blood isolates as well as reference OPA strains (n = 16) for their sensitivity to opsonophagocytosis and C3 deposition. Sixfold anticapsular antibody concentrations were required for 50% opsonophagocytic killing of 19F compared to that of 6B strains. Serotype 19F was more resistant to C3 deposition than 6B. Complement deposition and opsonophagocytosis were dependent on the concentration of anticapsular antibodies. Differences between pneumococcal serotypes in antibody-mediated protection may partly be explained by the abilities of the capsules to resist complement deposition. These findings support previous studies suggesting that higher antibody concentrations to the capsular polysaccharide are needed for protection against disease caused by serotype 19F than that caused by 6B.
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Jang, A.-Yeung, Min-Joo Choi, Yong Zhi, Hyun-Jung Ji, Ji-Yun Noh, Jin-Gu Yoon, Hee-Jin Cheong, Woo-Joo Kim, Ho-Seong Seo, and Joon-Young Song. "Development and Validation of Enzyme-Linked Immunosorbent Assay for Group B Streptococcal Polysaccharide Vaccine." Vaccines 9, no. 6 (May 21, 2021): 545. http://dx.doi.org/10.3390/vaccines9060545.
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Streptococcus agalactiae (group B Streptococcus, GBS) is a leading cause of neonatal sepsis and meningitis in infants. Limitations of prenatal GBS screening and intrapartum antibiotic prophylaxis render developing GBS vaccines a high priority. In this study, we developed an enzyme-linked immunosorbent assay (ELISA) for the practical and large-scale evaluation of GBS capsular polysaccharide (PS) vaccine immunogenicity against three main serotypes, Ia, III, and V. GBS-ELISA was developed and subsequently validated using a standardized curve-fitting four-parameter logistic method. Specificity was measured using adsorption of serum with homologous and heterologous PS. Homologous adsorption showed a ≥75% inhibition of all three serotypes, whereas with heterologous PS, IgG GBS-ELISA inhibited only ≤25% of serotypes III and V. However, with serotype Ia, IgG antibody levels decreased by >50%, even after adsorption with heterologous PS (III or V). In comparison, the inhibition opsonophagocytic killing assay (OPA) of serotypes Ia GBS exhibited a reduction in opsonophagocytic activity of only 20% and 1.1% for serotypes III and V GBS, respectively. The precision of the GBS-ELISA was assessed in five independent experiments using four serum samples. The coefficient of variation was <5% for all three serotypes. This standardized GBS-ELISA would be useful for GBS vaccine development and its evaluation.
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Hufnagel, Markus, Andrea Kropec, Christian Theilacker, and Johannes Huebner. "Naturally Acquired Antibodies against Four Enterococcus faecalis Capsular Polysaccharides in Healthy Human Sera." Clinical Diagnostic Laboratory Immunology 12, no. 8 (August 2005): 930–34. http://dx.doi.org/10.1128/cdli.12.8.930-934.2005.
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ABSTRACT Healthy human sera (HHS) contain naturally acquired enterococcal antibodies which promote neutrophil-mediated killing. The target antigens remain unknown. The present study used a capsular polysaccharide (CPS)-enzyme-linked immunosorbent assay (ELISA) to investigate whether the HHS antibodies of 12 healthy donors bound to the CPS of four E. faecalis serotypes (CPS-A to CPS-D) and then employed an opsonic-killing assay to determine if these antibodies mediated phagocyte-dependent killing. All HHS contained immunoglobulin G (IgG) and IgM antibodies directed against capsular polysaccharides of the four serotypes. Absorption of the sera with homologous and heterologous strains showed a majority of antibodies to be cross-reactive among the prototype strains. The susceptibility of the four prototype strains to opsonic killing varied. Opsonic killing of CPS-A and CPS-B strains was significantly higher than killing of CPS-C and CPS-D strains. Absorption studies revealed that the opsonic killing of HHS was only partially type specific, with cross-reactivity between CPS-A and CPS-B strains and between CPS-C and CPS-D strains. These data indicate that healthy individuals possess opsonic antibodies specific for CPS-A and CPS-B but only low titers of opsonic antibodies against CPS-C and CPS-D. Titers of opsonic antibodies did not correlate with antibody titers measured by ELISA. Whether this lack of correlation is due to the low frequency of opsonic antibodies or to increased resistance to the opsonophagocytic killing of some serotypes remains to be determined.
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Ledov, Vladimir A. "Functional and antigenspecific serum antibodies in mice after immunization with a candidate vaccine against Shigella flexneri 1b, 2a, 3a, 6, Y." Clinical Microbiology and Antimicrobial Chemotherapy 23, no. 4 (2021): 400–403. http://dx.doi.org/10.36488/cmac.2021.4.400-403.
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Objective. To determine functional anti-LPS specific serum bactericidal antibody (SBA) and opsonophagocytic killing antibody (OPKA) activities in mice immunized with a pentavalent candidate vaccine against Shigella flexneri 1b, 2a, 3a, 6, Y (PF). Materials and Methods. (CBA x C57 B1/6) F1 mice were immunized with a PF. 14 days after the reimmunization, serum samples were collected and the level of specific IgG in them was determined by ELISA. The sera of mice immunized with individual modified lipopolysaccharides (mLPS), which are part of PF, were used for positive control. Serum from intact mice served as a negative control. The functional activity of serum antibodies was determined by the methods of SBA and OPKA assay. The result was evaluated by the percentage of bacteria killed. Results. In all experimental groups of mice, an increase in the titer of specific IgG is observed (p < 0.05). The endpoint titer (ET) of anti-LPS S. flexneri 1b, 2a, 3a, 6, Y antibodies in the group of mice immunized with PF does not significantly differ from ET in the groups after immunizations with individual mLPS. At the same time, the indicators in the experimental groups are about 16 times higher than in the control. We determined a functional activity of S. flexneri-specific SBA and OPRA in mice immunized with PF. The rate of SBA killing was 54%, 66%, 35%, 60%, 60% for S. flexneri 1b, 2a, 3a, 6, Y serotypes, respectively. When OPKA killing in groups of immunized mice are 37%, 55%, 27%, 56%, 53% for S. flexneri 1b, 2a, 3a, 6, Y serotypes, respectively. Conclusions. PF induces the production of specific anti-LPS IgG comparable to its individual components. The sera from PF immunized mice contain functional antibodies. Serum bactericidal and opsonophagocytic assay are effective for use in a mouse model.
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Lin, Lin, Teclegiorgis Gebremariam, and Ashraf Ibrahim. "Antibodies targeting the Rhizopus oryzae invasin, CotH3, enhance neutrophil killing (VAC6P.952)." Journal of Immunology 192, no. 1_Supplement (May 1, 2014): 140.13. http://dx.doi.org/10.4049/jimmunol.192.supp.140.13.
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Abstract Mucormycosis is a lethal fungal infection that afflicts the immunocompromised host such as those suffering from hyperglycemia, ketoacidosis, other forms of acidosis, neutropenia, and those receiving corticosteroids. Rhizopus spp. are the most common pathogens isolated from patients with mucormycosis. Previously, we identified R. oryzae CotH3 as a surface protein which mediates fungal invasion of host cells during mucormycosis. We found rabbit polyclonal anti-CotH3 antibodies to significantly block host cell invasion by R. oryzae and protect mice from R. oryzae infection. However, it is unclear if CotH3 antibodies enhance phagocyte killing of the fungus. Here, we used flow cytometry and mouse neutrophil killing assays to determine the binding and opsonophagocytic killing capacities of varying concentrations of anti-CotH3 antibodies against R. oryzae, respectively. Anti-CotH3 antibodies showed high-affinity binding (>99%) to R. oryzae even when low concentration of the antibodies were used (e.g. 3mcg dose). CotH3 antibodies also widely bound to other Mucorales known to cause mucormycosis including Mucor circinelloides, Lichtheimia, Rhizomucor, and Cunninghamella. Neutrophil kill assay has shown Anti-CotH3 antibodies to dramatically enhance mouse neutrophil killing by > 2-fold increase vs. control with preimmune antibodies (P<0.04). Our studies warrant the further development of monoclonal antibodies targeting CotH3 protein as a novel therapy against mucormycosis.
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Riegler, Mann, Orihuela, and Tuomanen. "Opening the OPK Assay Gatekeeper: Harnessing Multi-Modal Protection by Pneumococcal Vaccines." Pathogens 8, no. 4 (October 23, 2019): 203. http://dx.doi.org/10.3390/pathogens8040203.
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Pneumococcal vaccine development is driven by the achievement of high activity in a single gatekeeper assay: the bacterial opsonophagocytic killing (OPK) assay. New evidence challenges the dogma that anti-capsular antibodies have only a single function that predicts success. The emerging concept of multi-modal protection presents an array of questions that are fundamental to adopting a new vaccine design process. If antibodies have hidden non-opsonic functions that are protective, should these be optimized for better vaccines? What would protein antigens add to protective activity? Are cellular immune functions additive to antibodies for success? Do different organs benefit from different modes of protection? Can vaccine activities beyond OPK protect the immunocompromised host? This commentary raises these issues at a time when capsule-only OPK assay-based vaccines are increasingly seen as a limiting strategy.
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Kampen, Annette H., Tore Tollersrud, and Arve Lund. "Staphylococcus aureus Capsular Polysaccharide Types 5 and 8 Reduce Killing by Bovine Neutrophils In Vitro." Infection and Immunity 73, no. 3 (March 2005): 1578–83. http://dx.doi.org/10.1128/iai.73.3.1578-1583.2005.
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ABSTRACT Isogenic variants of Staphylococcus aureus strain Reynolds expressing either no capsule or capsular polysaccharide (CP) type 5 (CP5) or type 8 (CP8) were used to assess the effect of CP on bacterial killing and the respiratory burst of bovine neutrophils. The effects of antisera specific for CP5 and CP8 were also evaluated. The killing of live bacteria by isolated neutrophils was quantified in a bactericidal assay, while the respiratory burst after stimulation with live bacteria in whole blood was measured by flow cytometry. The expression of a CP5 or CP8 capsule protected the bacteria from being killed by bovine neutrophils in vitro (P < 0.001), and the capsule-expressing variants did not stimulate respiratory burst activity in calf whole blood. The addition of serotype-specific antisera increased the killing of the capsule-expressing bacteria and enhanced their stimulating effect in the respiratory burst assay (P < 0.01). When the S. aureus variants were grown under conditions known not to promote capsule expression, there were no significant differences between them. The present study demonstrates that the expression of S. aureus CP5 or CP8 confers resistance to opsonophagocytic killing and prevents the bacteria from inducing respiratory burst of bovine neutrophils in vitro and that these effects can be reversed by the addition of serotype-specific antisera.
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Huebner, Johannes, Ying Wang, Wolfgang A. Krueger, Lawrence C. Madoff, Gayane Martirosian, Saskia Boisot, Donald A. Goldmann, Dennis L. Kasper, Arthur O. Tzianabos, and Gerald B. Pier. "Isolation and Chemical Characterization of a Capsular Polysaccharide Antigen Shared by Clinical Isolates ofEnterococcus faecalis and Vancomycin-ResistantEnterococcus faecium." Infection and Immunity 67, no. 3 (March 1, 1999): 1213–19. http://dx.doi.org/10.1128/iai.67.3.1213-1219.1999.
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ABSTRACT Enterococci are a common cause of serious infections, especially in newborns, severely immunocompromised patients, and patients requiring intensive care. To characterize enterococcal surface antigens that are targets of opsonic antibodies, rabbits were immunized with various gentamicin-killed Enterococcus faecalis strains, and immune sera were tested in an opsonophagocytic assay against a selection of clinical isolates. Serum raised against one strain killed the homologous strain (12030) at a dilution of 1:5,120 and mediated opsonic killing of 33% of all strains tested. In addition, this serum killed two (28%) of seven vancomycin-resistant Enterococcus faecium strains. Adsorption of sera with the homologous strain eliminated killing activity. The adsorbing antigens were resistant to treatment with proteinase K and to boiling for 1 h, but were susceptible to treatment with sodium periodate, indicating that the antigen inducing opsonic activity is a polysaccharide. Antibodies in immune rabbit sera reacted with a capsule-like structure visualized by electron microscopy both on the homologous E. faecalisstrain and on a vancomycin-resistant E. faecium strain. The capsular polysaccharides from E. faecalis 12030 andE. faecium 838970 were purified, and chemical and structural analyses indicated they were identical glycerol teichoic acid-like molecules with a carbohydrate backbone structure of 6-α-d-glucose-1-2 glycerol-3-PO4 with substitution on carbon 2 of the glucose with an α-2-1-d-glucose residue. The purified antigen adsorbed opsonic killing activity from immune rabbit sera and elicited high titers of antibodies (when used to immunize rabbits) that both mediated opsonic killing of bacteria and bound to a capsule-like structure visualized by electron microscopy. These results indicate that approximately one-third of a sample of 15 E. faecalisstrains and 7 vancomycin-resistant E. faecium strains possess shared capsular polysaccharides that are targets of opsonophagocytic antibodies and therefore are potential vaccine candidates.
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Brown, Martha, Rose Kowalski, Julie Zorman, Xin-min Wang, Victoria Towne, Qinjian Zhao, Susan Secore, et al. "Selection and Characterization of Murine Monoclonal Antibodies to Staphylococcus aureus Iron-Regulated Surface Determinant B with Functional Activity In Vitro and In Vivo." Clinical and Vaccine Immunology 16, no. 8 (June 24, 2009): 1095–104. http://dx.doi.org/10.1128/cvi.00085-09.
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ABSTRACT In an effort to characterize important epitopes of Staphylococcus aureus iron-regulated surface determinant B (IsdB), murine IsdB-specific monoclonal antibodies (MAbs) were isolated and characterized. A panel of 12 MAbs was isolated. All 12 MAbs recognized IsdB in enzyme-linked immunosorbent assays and Western blots; 10 recognized native IsdB expressed by S. aureus. The antigen epitope binding of eight of the MAbs was examined further. Three methods were used to assess binding diversity: MAb binding to IsdB muteins, pairwise binding to recombinant IsdB, and pairwise binding to IsdB-expressing bacteria. Data from these analyses indicated that MAbs could be grouped based on distinct or nonoverlapping epitope recognition. Also, MAb binding to recombinant IsdB required a significant portion of intact antigen, implying conformational epitope recognition. Four MAbs with nonoverlapping epitopes were evaluated for in vitro opsonophagocytic killing (OPK) activity and efficacy in murine challenge models. These were isotype switched from immunoglobulin G1 (IgG1) to IgG2b to potentially enhance activity; however, this isotype switch did not appear to enhance functional activity. MAb 2H2 exhibited OPK activity (≥50% killing in the in vitro OPK assay) and was protective in two lethal challenge models and a sublethal indwelling catheter model. MAb 13C7 did not exhibit OPK (<50% killing in the in vitro assay) and was protective in one lethal challenge model. Neither MAb 13G11 nor MAb 1G3 exhibited OPK activity in vitro or was active in a lethal challenge model. The data suggest that several nonoverlapping epitopes are recognized by the IsdB-specific MAbs, but not all of these epitopes induce protective antibodies.
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Humphries, Holly E., Charlotte Brookes, Lauren Allen, Eeva Kuisma, Andrew Gorringe, and Stephen Taylor. "Seroprevalence of Antibody-Mediated, Complement-Dependent Opsonophagocytic Activity against Neisseria meningitidis Serogroup B in England." Clinical and Vaccine Immunology 22, no. 5 (March 4, 2015): 503–9. http://dx.doi.org/10.1128/cvi.00100-15.
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ABSTRACTThe correlate of protection for the licensure of meningococcal vaccines is serum bactericidal activity. However, evidence indicates that a complex situation and other mechanisms, such as antibody-mediated, complement-dependent opsonophagocytosis (OP), may play a role in protection and should be investigated in order to understand immunity to this disease. In this study, a high-throughput flow cytometric opsonophagocytic assay (OPA) was optimized. The assay measures the presence of killed fluorescently labeledNeisseria meningitidiswithin human granulocytes (differentiated HL60 cells) by flow cytometry, using IgG-depleted pooled human plasma as an exogenous source of complement. This method was found to be reliable and correlated with the results of an opsonophagocytic killing assay. The OPA was used to measure OP activity in 1,878 serum samples from individuals ranging from 0 to 99 years of age againstN. meningitidisstrain NZ98/254 (B:4:P1.7-2,4). The levels of OP activity in individual serum samples varied greatly. OP activity showed an initial peak in the 6- to 12-month age group corresponding to a peak in disease incidence. The OP activity dropped in childhood until the late teenage years, although there was still a higher percentage of individuals with OP activity than with protective bactericidal antibody titers. OP activity reached a peak in the 30- to 39-year age group and then declined. This later peak in OP activity did not coincide with the young adults in whom peak serum bactericidal activity and disease incidence occurred. The demonstration of OP activity when disease incidence is low and when protective bactericidal antibody titers are not detected may indicate a role for OP in protection from meningococcal disease in these age groups.
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Burton, Robert L., and Moon H. Nahm. "Development and Validation of a Fourfold Multiplexed Opsonization Assay (MOPA4) for Pneumococcal Antibodies." Clinical and Vaccine Immunology 13, no. 9 (September 2006): 1004–9. http://dx.doi.org/10.1128/cvi.00112-06.
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ABSTRACT Opsonophagocytic killing assays (OPAs) are essential for developing and improving pneumococcal vaccines. There is a need for a high-throughput, reliable, standardized, and fully characterized OPA for pneumococcal antibodies. To meet the need, we have developed and characterized a fourfold multiplexed OPA (MOPA4) against 13 serotypes (1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F) of pneumococci. Thirteen target bacteria were made resistant to only one of the following antibiotics: optochin, streptomycin, spectinomycin, and trimethoprim. Following optimization of assay conditions, accuracy of MOPA4 was determined by testing 30 sera from old adults in the MOPA4 and the single-serotype assays. The opsonization titers obtained with both assays agreed well (r 2 > 0.95). Although 22 (out of 390; ∼6%) results differed more than twofold, the differences were not reproducible. The assay was specific: preabsorbing test sera with homologous polysaccharide (PS) completely abrogated opsonic activity, but a pool of unrelated PS (5 μg/ml of each) had no effect. Intra- and interassay coefficients of variation were 10 and 22%, respectively. MOPA4 results were unaffected by having different target pneumococcal serotypes in each assay group. Also, HL60 cell-to-bacteria ratios could be varied twofold without affecting the results. We conclude that MOPA4 is sensitive, accurate, specific, precise, and robust enough for large-scale clinical studies. Furthermore, MOPA4 should allow evaluation of multivalent pneumococcal vaccines with the limited volume of serum typically available from young children.
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Sharp, Julia A., Katelyn A. Cranmer, Megan G. Sage, and Keith L. Wycoff. "Complement Factor H Immunoproteins Increase Complement-mediated Opsonophagocytic Killing of Methicillin-resistant Staphylococcus aureus." Journal of Immunology 208, no. 1_Supplement (May 1, 2022): 181.02. http://dx.doi.org/10.4049/jimmunol.208.supp.181.02.
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Abstract Background As a major cause of a myriad of superficial and invasive infections worldwide, Staphylococcus aureus is a master of immune evasion, with the complement system being a primary target. Previously, we have shown that S. aureus surface protein SdrE binds the regulator Factor H (FH) to inhibit complement. Taking advantage of this interaction, we tested the effect of a fusion protein comprised of FH(18–20) fused to an IgG Fc on complement-mediated opsonophagocytosis and killing of methicillin resistant S. aureus. Methods Fusions were produced via the ΔXT/FT Nicotiana benthamiana plant expression system. Binding dynamics were examined via immunoblotting, fluorescence and ELISA. The effect of fusions on complement activation was determined by examining C3-fragment opsonization of S. aureus (total C3, C3b, iC3b) and subsequent C5a generation. The effect of fusions on S. aureus survival was assessed using an opsonphagocytosis assay with human polymorphonuclear cells. Results S. aureus bound significantly more FH-Fc compared to Fc-controls, and competed with serum FH for S. aureus binding. FH-Fc outperformed a complement inactive fusion variant in C3-fragment deposition and C5a generation, signifying an active role for the Fc region of FH-Fc. For 75% (3/4) of clinical isolates tested, FH-Fc significantly reduced S. aureus survival. Clinical isolates were all sdrE positive, sequence type 8, SCCmec IVa and Panton-Valentine Leukocidin (pvl) positive). Conclusion FH-Fc competitively inhibited serum FH from S. aureus binding and increased complement-mediated opsonophagocytosis and killing of CA-MRSA. Future studies will focus on enhancing the efficacy of FH-Fc fusion molecules as potential anti-staphylococcal therapeutics. Supported by a grant from the US Department of Defense, USAMRDC (W81XWH-19-1-0119).
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Givner, Laurence B. "Human Immunoglobulins for Intravenous Use: Comparison of Available Preparations for Group B Streptococcal Antibody Levels, Opsonic Activity, and Efficacy in Animal Models." Pediatrics 86, no. 6 (December 1, 1990): 955–62. http://dx.doi.org/10.1542/peds.86.6.955.
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Currently available human immunoglobulin preparations for intravenous use (IVIGs) are being used (with antibiotics) by some physicians for therapy of sepsis in newborns. Most neonatal sepsis and/or meningitis in this country is caused by group B Streptococcus (GBS), and most of these cases are due to type III GBS (III-GBS). The killing of III-GBS in vitro is dependent on specific IgG antibody. Adequate serum levels of specific III-GBS antibody protect the exposed newborn from the development of invasive disease. Therefore, III-GBS was used as a model to evaluate the activity of three IVIG preparations available for clinical use. Specific antibody levels, in vitro opsonophagocytic killing, and protective efficacy in animal models revealed differences in activity for III-GBS between the three IVIG preparations as well as between IVIG lots from the same manufacturer. Furthermore, it was found that the effect of IVIG using one of the assay methods may not reliably predict activity obtained using the other assays. These data document the inability to predict functional activity against a specific pathogen such as GBS on the part of a lot of IVIG chosen at random. In view of these findings and of the limited data evaluating clinical efficacy, IVIG cannot be recommended at this time for use in the therapy of infectious diseases such as neonatal sepsis.
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Tian, Haijun, Sarah Weber, Peter Thorkildson, Thomas R. Kozel, and Liise-anne Pirofski. "Efficacy of Opsonic and Nonopsonic Serotype 3 Pneumococcal Capsular Polysaccharide-Specific Monoclonal Antibodies against Intranasal Challenge with Streptococcus pneumoniae in Mice." Infection and Immunity 77, no. 4 (January 21, 2009): 1502–13. http://dx.doi.org/10.1128/iai.01075-08.
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ABSTRACT Serotype-specific antibodies to pneumococcal capsular polysaccharide (PPS) are a critical component of vaccine-mediated immunity to Streptococcus pneumoniae. In this study, we investigated the in vitro opsonophagocytic activities of three PPS-specific mouse immunoglobulin G1 monoclonal antibodies (MAbs), 1E2, 5F6, and 7A9, and determined their in vivo efficacies against intranasal challenge with WU2, a serotype 3 pneumococcal strain, in normal and immunodeficient mice. The MAbs had different in vitro activities in a pneumococcal killing assay: 7A9 enhanced killing by mouse neutrophils and J774 cells in the presence of a complement source, whereas 5F6 promoted killing in the absence, but not the presence, of complement, and 1E2 did not promote killing under any conditions. Nonetheless, all three MAbs protected normal and complement component 3-deficient mice from a lethal intranasal challenge with WU2 in passive-immunization experiments in which 10 μg of the MAbs were administered intraperitoneally before intranasal challenge. In contrast, only 1E2 protected Fcγ receptor IIB knockout (FcγRIIB KO) mice and mice that were depleted of neutrophils with the MAb RB6, whereas 7A9 and 5F6 required neutrophils and FcγRIIB to mediate protection. Conversely, 7A9 and 5F6 protected FcγR KO mice, but 1E2 did not. Hence, the efficacy of 1E2 required an activating FcγR(s), whereas 5F6 and 7A9 required the inhibitory FcγR (FcγRIIB). Taken together, our data demonstrate that both MAbs that do and do not promote pneumococcal killing in vitro can mediate protection in vivo, although their efficacies depend on different host receptors and/or components.
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Kropec, A., I. G. Sava, C. Vonend, T. Sakinc, E. Grohmann, and J. Huebner. "Identification of SagA as a novel vaccine target for the prevention of Enterococcus faecium infections." Microbiology 157, no. 12 (December 1, 2011): 3429–34. http://dx.doi.org/10.1099/mic.0.053207-0.
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Infections caused by multiresistant Gram-positive bacteria represent a major health burden in the community as well as in hospitalized patients. Enterococci, especially Enterococcus faecium, are well-known pathogens of hospitalized patients and are frequently linked with resistance against multiple antibiotics, which compromises effective therapy. Rabbit immune serum raised against heat-killed E. faecium E155, a HiRECC clone, was used in an opsonophagocytic assay, an inhibition assay and a mouse bacteraemia model to identify targets of opsonic and protective antibodies. Serum against whole heat-killed bacteria was opsonic and recognized a protein of about 72 kDa that was abundantly secreted. This protein, identified as SagA by LC-ES-MS/MS, was expressed in Escherichia coli and purified. Rabbit serum raised against the purified protein showed opsonic killing activity that was inhibited by almost 100 % using 100 µg purified protein ml−1. In a mouse bacteraemia model, a statistically significant reduction of the colony counts in blood was shown with immune rabbit serum compared with preimmune serum using the homologous and a heterologous vancomycin-resistant enterococci (VRE) strain. These results indicate that SagA could be used as a promising vaccine target to treat and/or prevent VRE bacteraemia.
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Martinez, Joseph E., Elizabeth A. Clutterbuck, Han Li, Sandra Romero-Steiner, and George M. Carlone. "Evaluation of Multiplex Flow Cytometric Opsonophagocytic Assays for Determination of Functional Anticapsular Antibodies to Streptococcus pneumoniae." Clinical and Vaccine Immunology 13, no. 4 (April 2006): 459–66. http://dx.doi.org/10.1128/cvi.13.4.459-466.2006.
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ABSTRACT The determination of functional antipneumococcal capsular polysaccharide antibodies by sequential testing of pre- and postvaccination serum samples one serotype at a time is sample-intensive and time-consuming and has a relatively low throughput. We tested several opsonophagocytic assay (OPA) formats, including the reference killing method, a monovalent bacterium-based flow method, a trivalent bacterium-based flow method, and a tetravalent bead-based flow method using a panel of sera (4 prevaccination and 16 postvaccination, from healthy adults immunized with the 23-valent pneumococcal polysaccharide vaccine). The trivalent and tetravalent methods allow simultaneous measurements of opsonic antibodies to multiple pneumococcal serotypes. The trivalent bacterial-flow OPA had significant correlation to the reference OPA method and to a previously published flow cytometric OPA (r values ranged from 0.61 to 0.91, P < 0.05) for serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F. The tetravalent OPA had significant correlation to all OPA method formats tested (r values from 0.68 to 0.92, P < 0.05) for all seven serotypes tested. This tetravalent OPA is an alternative to other OPA methods for use during vaccine evaluation and clinical trials. Further, the flow cytometric multiplex OPA format has the potential for expansion beyond the current four serotypes to eight or more serotypes, which would further increase relative sample throughput while reducing reagent and sample volumes used.
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Romero-Steiner, Sandra, Carl Frasch, Nelydia Concepcion, David Goldblatt, Helena Käyhty, Merja Väkeväinen, Craig Laferriere, et al. "Multilaboratory Evaluation of a Viability Assay for Measurement of Opsonophagocytic Antibodies Specific to the Capsular Polysaccharides of Streptococcus pneumoniae." Clinical Diagnostic Laboratory Immunology 10, no. 6 (November 2003): 1019–24. http://dx.doi.org/10.1128/cdli.10.6.1019-1024.2003.
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ABSTRACT Opsonophagocytosis is a correlate of protection that measures the functional activity of vaccine-induced antibodies. A standardized opsonophagocytosis assay (OPA) should be used as part of the evaluation of current and future pneumococcal (Pnc) polysaccharide (Ps)-based vaccines. We enrolled five laboratories to evaluate a previously standardized viability OPA. Each laboratory was provided with a detailed OPA protocol, seven target Pnc strains (serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F), two quality control sera and 12 paired sera (blinded) from adult donors who received one dose of the 23-valent Pnc Ps vaccine. Laboratories sent their results to the Centers for Disease Control and Prevention for analysis. Sera were tested in duplicate (single run), and the results were averaged to yield a single OPA titer (≥50% killing) for each serum sample. The percentage of sera within one or two dilutions of the calculated median OPA titer was determined for each laboratory and for each serotype. In general, laboratories were capable of detecting OPA titers within one or two dilutions of the median for at least 75 and 88%, respectively, of the sera tested. The level of agreement with the median OPA titers varied depending on the participating laboratory (overall agreement = 0.8 [99% confidence interval = 0.75 to 0.85]). All OPA median titers reported for quality control sera were within one dilution of the expected titer. We conclude that this OPA can be done in multiple laboratories with a high degree of interlaboratory reproducibility.
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Held, Thomas K., Nina R. M. Jendrike, Tomislav Rukavina, Rainer Podschun, and Matthias Trautmann. "Binding to and Opsonophagocytic Activity of O-Antigen-Specific Monoclonal Antibodies against Encapsulated and NonencapsulatedKlebsiella pneumoniae Serotype O1 Strains." Infection and Immunity 68, no. 5 (May 1, 2000): 2402–9. http://dx.doi.org/10.1128/iai.68.5.2402-2409.2000.
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ABSTRACT The high mortality of nosocomial infections caused byKlebsiella spp. has acted as a stimulus to develop immunotherapeutic approaches targeted against surface molecules of these bacteria. Since O-antigen-specific antibodies may add to the protective effect of K antisera, we tested the functional and binding capacity of O-antigen-specific monoclonal antibodies (MAbs) raised against different Klebsiella O antigens. The MAbs tested were specific for the O-polysaccharide partial antigensd-galactan II (MAb Ru-O1), d-galactan I (MAb IV/4-5), or core oligosaccharide (MAb V/9-5) of theKlebsiella serogroup O1 antigen. In enzyme-linked immunosorbent assay binding experiments, we found that all MAbs recognized their epitopes on intact capsule-free bacteria; however, binding to encapsulated wild-type strains belonging to different K-antigen serotypes was significantly reduced. The K2 antigen acted as the strongest penetration barrier, while the K7 and K21 antigens allowed some, though diminished, antibody binding. In vitro phagocytic killing experiments showed that MAb Ru-O1 possessed significant opsonizing activity for nonencapsulated O1 serogroup strains and also, to a much lesser extent, for encapsulated strains belonging to the O1:K7 and O1:K21 serotypes. MAbs or antisera specific for thed-galactan II antigen may thus be the most promising agents for further efforts to develop a second-generationKlebsiella hyperimmune globulin comprising both K- and O-antigen specificities.
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Choi, Eun Hwa, Fan Zhang, Ying-Jie Lu, and Richard Malley. "Capsular Polysaccharide (CPS) Release by Serotype 3 Pneumococcal Strains Reduces the Protective Effect of Anti-Type 3 CPS Antibodies." Clinical and Vaccine Immunology 23, no. 2 (December 16, 2015): 162–67. http://dx.doi.org/10.1128/cvi.00591-15.
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ABSTRACTThe efficacy of the serotype 3 (ST3) pneumococcal conjugate vaccine (PCV) remains unclear. While the synthesis of capsular polysaccharide (CPS) of most serotypes iswzydependent, the strains of two serotypes, 3 and 37, synthesize CPS by the synthase-dependent pathway, resulting in a polysaccharide that is not covalently linked to peptidoglycan and can be released during growth. We hypothesized that the release of CPS during growth reduces anti-type 3 CPS antibody-mediated protection and may explain the lower efficacy of the type 3 component of PCV than that of other PCVs. Thein vitro-released CPS concentrations per 107CFU of ST3 and ST37 strains were significantly higher than those for the ST1, ST4, ST6B, and ST14 strains. Following intraperitoneal (i.p.) injection in mice, blood concentrations of CPS were significantly higher for the ST3 than for the ST4/5 strains. The opsonophagocytic killing assay (OPKA) titer of anti-type 3 CPS antibody was significantly reduced by type 3 CPS, culture supernatant, or serum fromStreptococcus pneumoniaeST3 strain WU2-infected mice. Mice were injected with capsule-specific antibodies and challenged i.p. with or without the addition of sterile culture supernatant containing type-specific CPS. The addition of 0.2 μl of culture supernatant from WU2 inhibited passive protection, whereas 100-fold-more culture supernatant fromS. pneumoniaeST4 strain TIGR4 was required for the inhibition of protection. We conclude that released type 3 CPS interferes with antibody-mediated killing and protection by anti-CPS antibodies. The relative failure of ST3 PCV may be due to CPS release, suggesting that alternative immunization approaches for ST3 may be necessary.
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Burns, Tamika, Zhaojing Zhong, Michael Steinitz, and Liise-anne Pirofski. "Modulation of Polymorphonuclear Cell Interleukin-8 Secretion by Human Monoclonal Antibodies to Type 8 Pneumococcal Capsular Polysaccharide." Infection and Immunity 71, no. 12 (December 2003): 6775–83. http://dx.doi.org/10.1128/iai.71.12.6775-6783.2003.
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ABSTRACT Pneumococcal capsular polysaccharide (PS) vaccines induce type-specific immunoglobulin M (IgM), IgG, and IgA. Type-specific IgG to the PS is sufficient to confer protection against the homologous serotype of the pneumococcus, but the efficacies of type-specific IgM and IgA are less well understood. We examined the in vitro activities and efficacies in mice of two human monoclonal antibodies (MAbs) to type 8 PS, NAD (IgA) and D11 (IgM). MAb-mediated opsonophagocytic killing was evaluated after coculture of type 8 pneumococci with human polymorphonuclear cells (PMNs), type-specific or control MAbs, and human complement sources. The effects of the MAbs on PMN interleukin-8 (IL-8) and IL-6 secretion were determined in supernatants from cocultures containing pneumococci and PMNs by enzyme-linked immunosorbent assay. MAb efficacy was determined in an intratracheal model of type 8 infection in mice with classical complement pathway deficiency. Both MAbs were protective in 100% of infected mice. Neither MAb promoted a significant amount of killing of type 8 pneumococci compared to its isotype control MAb. Both type-specific MAbs mediated complement-dependent modulation of PMN IL-8 secretion, with increased secretion at effector/target (E:T) ratios of 500:1 and 50:1 and reduced secretion at 1:5. Trypan blue staining revealed that PMNs cocultured with D11 were less viable at an E:T ratio of 1:5 than PMNs cocultured with the control MAb. PMN IL-6 secretion was increased by both type-specific and control MAbs. These results suggest that certain type-specific IgM and IgAs might contribute to host defense by modulation of the inflammatory response to pneumococci.
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Caro-Aguilar, Ivette, Elizabeth Ottinger, Robert Hepler, Deborah Nahas, Chengwei Wu, Joseph Joyce, Jon Heinrichs, and Julie Skinner. "Immunogenicity of a Group A Streptococcal peptide vaccine conjugated to CRM197 (53.12)." Journal of Immunology 186, no. 1_Supplement (April 1, 2011): 53.12. http://dx.doi.org/10.4049/jimmunol.186.supp.53.12.
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Abstract Group A Streptococcus (GAS) causes several disease manifestations and remains a huge medical burden in developing and developed countries, therefore there is an imminent need for a prophylactic vaccine. One focal point of vaccine development for GAS has been the M protein, an alpha-helical coiled-coil surface protein. An epitope derived from the C-terminal region of the M protein, J8, has been shown to be highly immunogenic and to generate opsonophagocytic killing (OPK) activity and protection in mice. Here we evaluated the use of an alternative protein carrier, CRM197, a molecularly detoxified version of diphtheria toxoid (DT), by conjugating J8 to CRM197 and immunizing C3H and Balb/c mice with this vaccine on AAHSA (Amorphous Aluminum Hydroxyphosphate Sulfate Adjuvant). J8-specific ELISA and whole bacteria immunofluorescence (IFA) were used to evaluate immunogenicity. Functional antibody activity was evaluated by opsonophagocytosis (OPK) and protection from a GAS intranasal challenge. J8 conjugated to CRM197 induced high antibody responses to J8; these specific antibodies recognized the epitope on the surface of the bacteria by IFA and demonstrated functional activity in an OPK assay. Additionally, J8-CRM197 immunized mice demonstrated significantly greater survival than adjuvant control immunized mice. This study showed that CRM197 is a suitable carrier protein for J8 peptide conjugation in C3H and Balb/c mice.
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Khan, Naeem, Raies Ahmad Qadri, and Devinder Sehgal. "Correlation betweenIn VitroComplement Deposition and Passive Mouse Protection of Anti-Pneumococcal Surface Protein A Monoclonal Antibodies." Clinical and Vaccine Immunology 22, no. 1 (November 19, 2014): 99–107. http://dx.doi.org/10.1128/cvi.00001-14.
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ABSTRACTThe shortcomings of the licensed polysaccharide-based pneumococcal vaccine are driving efforts toward development of a protein-based vaccine that is serotype independent and effective in all age groups. An opsonophagocytic killing assay (OPKA) is used to evaluate the antibody response against polysaccharide-based pneumococcal vaccines. However, the OPKA is not reliable for noncapsular antigens. Thus, there is a need to develop anin vitrosurrogate for protection for protein vaccine candidates like pneumococcal surface antigen A (PspA). PspA is a serologically variable cell surface virulence factor. Based on its sequence, PspA has been classified into families 1 (clade 1 and 2), 2 (clades 3, 4 and 5), and 3 (clade 6). Here, we report the characterization of 18 IgG anti-PspA monoclonal antibodies (anti-PspAhkR36AMAbs) generated from mice immunized with heat-killed strain R36A (clade 2). An enzyme-linked immunosorbent assay (ELISA)-based analysis of the reactivity of the MAbs with recombinant PspAs from the 6 clades indicated that they were family 1 specific. This was confirmed by flow cytometry using a hyperimmune serum generated against PspA from R36A. Eight MAbs that bind at least one clade 1- and clade 2-expressing strain were evaluated for complement deposition, bactericidal activity, and passive protection. The anti-PspAhkR36AMAb-dependent deposition of complement on pneumococci showed a positive correlation with passive protection against strain WU2 (r= 0.8783,P= 0.0041). All of our protective MAbs showed bactericidal activity; however, not all MAbs that exhibited bactericidal activity conferred protectionin vivo. The protective MAbs described here can be used to identify conserved protection eliciting B cell epitopes for engineering a superior PspA-based vaccine.
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Guttormsen, Hilde-Kari, Carol J. Baker, Moon H. Nahm, Lawrence C. Paoletti, Susu M. Zughaier, Morven S. Edwards, and Dennis L. Kasper. "Type III Group B Streptococcal Polysaccharide Induces Antibodies That Cross-React with Streptococcus pneumoniae Type 14." Infection and Immunity 70, no. 4 (April 2002): 1724–38. http://dx.doi.org/10.1128/iai.70.4.1724-1738.2002.
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ABSTRACT Covalent linkage of a bacterial polysaccharide to a protein greatly enhances the carbohydrate's immunogenicity and its binding to solid surfaces in immunoassays. These findings have spurred the development of glycoconjugate vaccines to prevent serious bacterial infections as well as the use of glycoconjugates as coating antigens in bioassays. We evaluated sera from women immunized with unconjugated group B streptococcal (GBS) type III (GBS III) polysaccharide (IIIPS) or with IIIPS covalently linked to tetanus toxoid to assess specificity, sensitivity, and parallelism in dilution curves in two GBS III enzyme-linked immunosorbent assays (ELISAs). One assay used IIIPS mixed with methylated human serum albumin (IIIPS + mHSA) as the coating antigen, and the other used IIIPS covalently linked to HSA (III-HSA). Each coating antigen was associated with a highly specific GBS III bioassay. The sensitivity was higher in the III-HSA ELISA, in which conjugated IIIPS is bound to the plates. Parallelism in titration curves was observed in the III-HSA but not in the IIIPS + mHSA ELISA. The excellent correlation between the concentrations of GBS IIIPS-specific immunoglobulin G (IgG) and the opsonophagocytic activity of these antibodies indicated that the III-HSA assay can predict functionality of vaccine-induced IgG against GBS III disease. The structure of the repeating unit of the capsular polysaccharide of GBS III differs from that of Streptococcus pneumoniae type 14 (Pn14 PS) only by the presence on GBS III of a sialic acid residue at the end of the side chain. The majority of healthy adults responding to GBS III vaccines with a fourfold or greater increase in GBS III-specific IgG antibodies developed antibodies cross-reacting with Pn14 PS (i.e., desialylated GBS IIIPS). The proportion of GBS vaccine responders who developed IgG to the desialylated IIIPS did not depend on whether IIIPS was given in the unconjugated or conjugated form. When present, these vaccine-induced cross-reacting antibodies conferred in vitro antibody-mediated opsonophagocytosis and killing of both GBS III and Pn14, two pathogens that cause invasive disease in young infants.
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Mathews, Christine E., Eric L. Brown, Perla J. Martinez, Upasana Bagaria, Moon H. Nahm, Robert L. Burton, Susan P. Fisher-Hoch, Joseph B. McCormick, and Shaper Mirza. "Impaired Function of Antibodies to Pneumococcal Surface Protein A but Not to Capsular Polysaccharide in Mexican American Adults with Type 2 Diabetes Mellitus." Clinical and Vaccine Immunology 19, no. 9 (July 3, 2012): 1360–69. http://dx.doi.org/10.1128/cvi.00268-12.
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ABSTRACTThe goal of the study was to determine baseline protective titers of antibodies toStreptococcus pneumoniaesurface protein A (PspA) and capsular polysaccharide in individuals with and individuals without type 2 diabetes mellitus. A total of 561 individuals (131 individuals with diabetes and 491 without) were screened for antibodies to PspA using a standard enzyme-linked immunosorbent assay (ELISA). A subset of participants with antibodies to PspA were retested using a WHO ELISA to determine titers of antibodies to capsular polysaccharide (CPS) (serotypes 4, 6B, 9V, 14, 18C, 19A, 19F, and 23F). Functional activity of antibodies was measured by assessing their ability to enhance complement (C3) deposition on pneumococci and promote killing of opsonized pneumococci. Titers of antibodies to protein antigens (PspA) were significantly lower in individuals with diabetes than controls without diabetes (P= 0.01), and antibodies showed a significantly reduced complement deposition ability (P= 0.02). Both antibody titers and complement deposition were negatively associated with hyperglycemia. Conversely, titers of antibodies to capsular polysaccharides were either comparable between the two groups or were significantly higher in individuals with diabetes, as was observed for CPS 14 (P= 0.05). The plasma specimens from individuals with diabetes also demonstrated a higher opsonophagocytic index against CPS serotype 14. Although we demonstrate comparable protective titers of antibodies to CPS in individuals with and individuals without diabetes, those with diabetes had lower PspA titers and poor opsonic activity strongly associated with hyperglycemia. These results suggest a link between diabetes and impairment of antibody response.
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Theilacker, Christian, Zbigniew Kaczynski, Andrea Kropec, Francesca Fabretti, Tatjana Sange, Otto Holst, and Johannes Huebner. "Opsonic Antibodies to Enterococcus faecalis Strain 12030 Are Directed against Lipoteichoic Acid." Infection and Immunity 74, no. 10 (October 2006): 5703–12. http://dx.doi.org/10.1128/iai.00570-06.
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ABSTRACT A teichoic acid (TA)-like polysaccharide in Enterococcus faecalis has previously been shown to induce opsonic antibodies that protect against bacteremia after active and passive immunization. Here we present new data providing a corrected structure of the antigen and the epitope against which the opsonic antibodies are directed. Capsular polysaccharide isolated from E. faecalis strain 12030 by enzymatic digestion of peptidoglycan and chromatography (enzyme-TA) was compared with lipoteichoic acid (LTA) extracted using butanol and purified by hydrophobic-interaction chromatography (BuOH-LTA). Structural determinations were carried out by chemical analysis and nuclear magnetic resonance spectroscopy. Antibody specificity was assessed by enzyme-linked immunosorbent assay and the opsonophagocytosis assay. After alanine ester hydrolysis, there was structural identity between enzyme-TA and BuOH-LTA of the TA-parts of the two molecules. The basic enterococcal LTA structure was confirmed: 1,3-poly(glycerol phosphate) nonstoichiometrically substituted at position C-2 of the glycerol residues with d-Ala and kojibiose. We also detected a novel substituent at position C-2, [d-Ala→6]-α-d-Glcp-(1→2-[d-Ala→6]-α-d-Glcp-1→). Antiserum raised against enzyme-TA bound equally well to BuOH-LTA and dealanylated BuOH-LTA as to the originally described enzyme-TA antigen. BuOH-LTA was a potent inhibitor of opsonophagocytic killing by the antiserum to enzyme-TA. Immunization with antibiotic-killed whole bacterial cells did not induce a significant proportion of antibodies directed against alanylated epitopes on the TA, and opsonic activity was inhibited completely by both alanylated and dealanylated BuOH-LTA. In summary, the E. faecalis strain 12030 enzyme-TA is structurally and immunologically identical to dealanylated LTA. Opsonic antibodies to E. faecalis 12030 are directed predominantly to nonalanylated epitopes on the LTA molecule.
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Li, Yuanyi, Marcelo Gottschalk, Miriam Esgleas, Sonia Lacouture, J. Daniel Dubreuil, Philip Willson, and Josee Harel. "Immunization with Recombinant Sao Protein Confers Protection against Streptococcus suis Infection." Clinical and Vaccine Immunology 14, no. 8 (June 13, 2007): 937–43. http://dx.doi.org/10.1128/cvi.00046-07.
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ABSTRACT Sao is a Streptococcus suis surface protein recently identified as a potential vaccine candidate. In this study, recombinant Sao in combination with Quil A provided cross-protection against S. suis serotype 2 disease in mouse and pig vaccination protocols. Subcutaneous immunization of mice elicited strong immunoglobulin G (IgG) antibody responses. All four IgG subclasses were induced, with the IgG2a titer being the highest, followed by those of IgG1, IgG2b, and IgG3. Challenge of the mice with S. suis strain 31533 resulted in a mortality rate of 80% for the control group, which received Quil A only. In contrast, all of the mice immunized with Sao survived. In a pig vaccination protocol, intramuscular immunization with Sao also elicited significant humoral antibody responses, and both the IgG1 and IgG2 subclasses were induced, with a predominance of IgG2 production. In vitro assay showed that Sao-induced antibodies significantly promoted the ability of porcine neutrophils in opsonophagocytic killing of S. suis. An aerosol challenge of the pigs with S. suis strain 166 resulted in clinical signs characteristic of S. suis infection in diseased pigs. The vaccine group showed significantly better survival, lower clinical scores, and less S. suis recovery from postmortem tissue samples than did the control group. Furthermore, this study also revealed that although challenge S. suis strains express Sao size variants, recombinant Sao conferred cross-protection. These data demonstrate that recombinant Sao formulated with Quil A triggers strong opsonizing antibody responses which confer efficient immunity against challenge infection with heterologous S. suis type 2.
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Herring, Sydney E., Sovathiro Mao, Manmeet Bhalla, Essi Y. I. Tchalla, Jill M. Kramer, and Elsa N. Bou Ghanem. "Mitochondrial ROS production by neutrophils is required for host antimicrobial function against Streptococcus pneumoniae and is controlled by A2B adenosine receptor signaling." PLOS Pathogens 18, no. 11 (November 14, 2022): e1010700. http://dx.doi.org/10.1371/journal.ppat.1010700.
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Polymorphonuclear cells (PMNs) control Streptococcus pneumoniae (pneumococcus) infection through various antimicrobial activities. We previously found that reactive oxygen species (ROS) were required for optimal antibacterial function, however, the NADPH oxidase is known to be dispensable for the ability of PMNs to kill pneumococci. In this study, we explored the role of ROS produced by the mitochondria in PMN antimicrobial defense against pneumococci. We found that the mitochondria are an important source of overall intracellular ROS produced by murine PMNs in response to infection. We investigated the host and bacterial factors involved and found that mitochondrial ROS (MitROS) are produced independent of bacterial capsule or pneumolysin but presence of live bacteria that are in direct contact with PMNs enhanced the response. We further found that MyD88-/- PMNs produced less MitROS in response to pneumococcal infection suggesting that released bacterial products acting as TLR ligands are sufficient for inducing MitROS production in PMNs. To test the role of MitROS in PMN function, we used an opsonophagocytic killing assay and found that MitROS were required for the ability of PMNs to kill pneumococci. We then investigated the role of MitROS in host resistance and found that MitROS are produced by PMNs in response to pneumococcal infection. Importantly, treatment of mice with a MitROS scavenger prior to systemic challenge resulted in reduced survival of infected hosts. In exploring host pathways that control MitROS, we focused on extracellular adenosine, which is known to control PMN anti-pneumococcal activity, and found that signaling through the A2B adenosine receptor inhibits MitROS production by PMNs. A2BR-/- mice produced more MitROS and were significantly more resistant to infection. Finally, we verified the clinical relevance of our findings using human PMNs. In summary, we identified a novel pathway that controls MitROS production by PMNs, shaping host resistance against S. pneumoniae.
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Dalia, Ankur B., Alistair J. Standish, and Jeffrey N. Weiser. "Three Surface Exoglycosidases from Streptococcus pneumoniae, NanA, BgaA, and StrH, Promote Resistance to Opsonophagocytic Killing by Human Neutrophils." Infection and Immunity 78, no. 5 (February 16, 2010): 2108–16. http://dx.doi.org/10.1128/iai.01125-09.
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ABSTRACT Streptococcus pneumoniae (the pneumococcus) is a major human pathogen and a leading cause of inflammatory infections such as pneumonia and otitis media. An important mechanism for host defense against S. pneumoniae is opsonophagocytic killing by neutrophils. To persist in the human host, the pneumococcus has developed strategies to evade opsonization and subsequent neutrophil-mediated killing. Utilizing a genomic approach, we identified NanA, the major pneumococcal neuraminidase, as a factor important for resistance to opsonophagocytic killing in ex vivo killing assays using human neutrophils. The effect of NanA was shown using both type 4 (TIGR4) and type 6A clinical isolates. NanA promotes this resistance by acting in conjunction with two other surface-associated exoglycosidases, BgaA, a β-galactosidase, and StrH, an N-acetylglucosaminidase. Experiments using human serum showed that these exoglycosidases reduced deposition of complement component C3 on the pneumococcal surface, providing a mechanism for this resistance. Additionally, we have shown that antibodies in human serum do not contribute to this phenotype. These results demonstrate that deglycosylation of a human serum glycoconjugate(s) by the combined effects of NanA, BgaA, and StrH, is important for resistance to complement deposition and subsequent phagocytic killing of S. pneumoniae.
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Thakker, Manoj, Jin-Sir Park, Vincent Carey, and Jean C. Lee. "Staphylococcus aureus Serotype 5 Capsular Polysaccharide Is Antiphagocytic and Enhances Bacterial Virulence in a Murine Bacteremia Model." Infection and Immunity 66, no. 11 (November 1, 1998): 5183–89. http://dx.doi.org/10.1128/iai.66.11.5183-5189.1998.
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ABSTRACT Controversy persists over the role that the capsular polysaccharide plays in the pathogenesis of Staphylococcus aureusinfections. To address this issue, we compared the mouse virulence ofS. aureus Reynolds and capsule-defective mutant strains cultivated under conditions of high or low capsule expression. Strain Reynolds cells cultivated on Columbia salt agar plates expressed ∼100-fold more type 5 capsular polysaccharide than did cells cultivated in Columbia salt broth. The relative virulence of strain Reynolds and its capsule-defective mutants after growth on either solid or liquid medium was examined in mice challenged intraperitoneally or intravenously. The results indicated that agar-grown Reynolds cells were cleared from the bloodstream of mice less readily than broth-grown Reynolds cells. When the parental and mutant strains were cultivated on solid medium, strain Reynolds sustained a higher level of bacteremia than did the capsular mutants. We performed in vitro opsonophagocytic killing assays to determine whether staphylococcal virulence for mice correlated with resistance to phagocytosis. S. aureus Reynolds cultivated on solid medium was susceptible to phagocytic killing only in the presence of specific capsular antibodies and complement. Strain Reynolds grown in broth showed opsonic requirements for phagocytic killing that were similar to those of the capsular mutants (grown in broth or on agar); i.e., the bacteria were opsonized for phagocytosis by nonimmune serum with complement activity. These studies indicate that optimal expression of capsule enhances bacterial virulence in the mouse model of bacteremia, probably by rendering the organisms resistant to opsonophagocytic killing by leukocytes.
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Watts, Andrew, Danbing Ke, Qun Wang, Anil Pillay, Anne Nicholson-Weller, and Jean C. Lee. "Staphylococcus aureus Strains That Express Serotype 5 or Serotype 8 Capsular Polysaccharides Differ in Virulence." Infection and Immunity 73, no. 6 (June 2005): 3502–11. http://dx.doi.org/10.1128/iai.73.6.3502-3511.2005.
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ABSTRACT Most isolates of Staphylococcus aureus produce a serotype 5 (CP5) or 8 (CP8) capsular polysaccharide. To investigate whether CP5 and CP8 differ in their biological properties, we created isogenic mutants of S. aureus Reynolds that expressed CP5, CP8, or no capsule. Biochemical analyses of CP5 and CP8 purified from the isogenic S. aureus strains were consistent with published structures. The degree of O acetylation of each polysaccharide was similar, but CP5 showed a greater degree of N acetylation. Mice challenged with the CP5+ strain showed a significantly higher bacteremia level than mice challenged with the CP8+ strain. Similarly, the CP5+ strain survived preferentially in the bloodstream and kidneys of infected mice challenged with a mixed inoculum containing both strains. The enhanced virulence of the CP5+ strain in vivo correlated with its greater resistance to in vitro killing in whole mouse blood. Likewise, in vitro opsonophagocytic killing assays with human neutrophils and sera revealed greater survival of the Reynolds (CP5) strain, even though the kinetics of opsonization by C3b and iC3b was similar for both the CP5+ and CP8+ strains. Electron micrographs demonstrated C3 molecules on the cell wall beneath the capsule layer for both serotype 5 and 8 strains. Purified CP5 and CP8 stimulated a modest oxidative burst in human neutrophils but failed to activate the alternative complement pathway. These results indicate that CP5 and CP8 differ in a number of biological properties, and these differences likely contribute to the relative virulence of serotype 5 and 8 S. aureus in vivo.
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Romero-Saavedra, F., D. Laverde, E. Kalfopoulou, C. Martini, R. Torelli, D. Martinez-Matamoros, M. Sanguinetti, and J. Huebner. "Conjugation of Different Immunogenic Enterococcal Vaccine Target Antigens Leads to Extended Strain Coverage." Journal of Infectious Diseases 220, no. 10 (July 9, 2019): 1589–98. http://dx.doi.org/10.1093/infdis/jiz357.
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Abstract Enterococci have emerged as important nosocomial pathogens due to their resistance to the most commonly used antibiotics. Alternative treatments or prevention options are aimed at polysaccharides and surface-related proteins that play important roles in pathogenesis. Previously, we have shown that 2 Enterococcus faecium proteins, the secreted antigen A and the peptidyl-prolyl cis-trans isomerase, as well as the Enterococcus faecalis polysaccharide diheteroglycan, are able to induce opsonic and cross-protective antibodies. Here, we evaluate the use of glycoconjugates consisting of these proteins and an enterococcal polysaccharide to develop a vaccine with broader strain coverage. Diheteroglycan was conjugated to these 2 enterococcal proteins. Rabbit sera raised against these glycoconjugates showed Immunoglobulin G titers against the corresponding conjugate, as well as against the respective protein and carbohydrate antigens. Effective opsonophagocytic killing for the 2 sera was observed against different E. faecalis and E. faecium strains. Enzyme-linked immunosorbent assays against whole bacterial cells showed immune recognition of 22 enterococcal strains by the sera. Moreover, the sera conferred protection against E. faecalis and E. faecium strains in a mouse infection model. Our results suggest that these glycoconjugates are promising candidates for vaccine formulations with a broader coverage against these nosocomial pathogens and that the evaluated proteins are potential carrier proteins.
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DiGiandomenico, Antonio, Paul Warrener, Melissa Hamilton, Sandrine Guillard, Peter Ravn, Ralph Minter, Maria Margarita Camara, et al. "Identification of broadly protective human antibodies to Pseudomonas aeruginosa exopolysaccharide Psl by phenotypic screening." Journal of Experimental Medicine 209, no. 7 (June 25, 2012): 1273–87. http://dx.doi.org/10.1084/jem.20120033.
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Pseudomonas aeruginosa is a leading cause of hospital-associated infections in the seriously ill, and the primary agent of chronic lung infections in cystic fibrosis patients. A major obstacle to effective control of P. aeruginosa infections is its intrinsic resistance to most antibiotic classes, which results from chromosomally encoded drug-efflux systems and multiple acquired resistance mechanisms selected by years of aggressive antibiotic therapy. These factors demand new strategies and drugs to prevent and treat P. aeruginosa infections. Herein, we describe a monoclonal antibody (mAb) selection strategy on whole P. aeruginosa cells using single-chain variable fragment phage libraries derived from healthy individuals and patients convalescing from P. aeruginosa infections. This approach enabled identification of mAbs that bind three distinct epitopes on the product of the Psl. This exopolysaccharide is important for P. aeruginosa attachment to mammalian cells, and for the formation and maintenance of biofilms produced by nonmucoid and mucoid P. aeruginosa isolates. Functional screens revealed that mAbs to one epitope exhibit superior activity in opsonophagocytic killing and cell attachment assays, and confer significant protection in multiple animal models. Our results indicate that Psl is an accessible serotype-independent surface feature and promising novel protective antigen for preventing P. aeruginosa infections. Furthermore, our mAb discovery strategy holds promise for application to other bacterial pathogens.
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Giefing, Carmen, Andreas L. Meinke, Markus Hanner, Tamás Henics, Duc Bui Minh, Dieter Gelbmann, Urban Lundberg, et al. "Discovery of a novel class of highly conserved vaccine antigens using genomic scale antigenic fingerprinting of pneumococcus with human antibodies." Journal of Experimental Medicine 205, no. 1 (December 31, 2007): 117–31. http://dx.doi.org/10.1084/jem.20071168.
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Pneumococcus is one of the most important human pathogens that causes life-threatening invasive diseases, especially at the extremities of age. Capsular polysaccharides (CPSs) are known to induce protective antibodies; however, it is not feasible to develop CPS-based vaccines that cover all of the 90 disease-causing serotypes. We applied a genomic approach and described the antibody repertoire for pneumococcal proteins using display libraries expressing 15–150 amino acid fragments of the pathogen's proteome. Serum antibodies of exposed, but not infected, individuals and convalescing patients identified the ANTIGENome of pneumococcus consisting of ∼140 antigens, many of them surface exposed. Based on several in vitro assays, 18 novel candidates were preselected for animal studies, and 4 of them showed significant protection against lethal sepsis. Two lead vaccine candidates, protein required for cell wall separation of group B streptococcus (PcsB) and serine/threonine protein kinase (StkP), were found to be exceptionally conserved among clinical isolates (>99.5% identity) and cross-protective against four different serotypes in lethal sepsis and pneumonia models, and have important nonredundant functions in bacterial multiplication based on gene deletion studies. We describe for the first time opsonophagocytic killing activity for pneumococcal protein antigens. A vaccine containing PcsB and StkP is intended for the prevention of infections caused by all serotypes of pneumococcus in the elderly and in children.