Group B streptococcus (GBS) infection is a leading cause of sepsis and meningitis among infants, and is associated with high rates of morbidity and mortality in many countries. Protection against GBS typically involves antibody-mediated opsonization by phagocytes and complement components. The present study evaluated serotype-specific functional antibodies to GBS among Korean infants and in intravenous immunoglobulin (IVIG) products. An opsonophagocytic killing assay (OPA) was used to calculate the opsonization indices (OIs) of functional antibodies to serotypes Ia, Ib, and III in 19 IVIG products from 5 international manufacturers and among 98 Korean infants (age: 0–11 months). The GBS Ia, Ib, and III serotypes were selected because they are included in a trivalent GBS vaccine formulation that is being developed. The OI values for the IVIG products were 635–5,706 (serotype Ia), 488–1,421 (serotype Ib), and 962–3,315 (serotype III), and none of the IVIG lots exhibited undetectable OI values (< 4). The geometric mean OI values were similar for all 3 serotypes when we compared the Korean manufacturers. The seropositive rate among infants was significantly lower for serotype Ia (18.4%), compared to serotype Ib and serotype III (both, 38.8%). Infant age of ≥ 3 months was positively correlated with the seropositive rates for each serotype. Therefore, only a limited proportion of infants exhibited protective immunity against serotype Ia, Ib, and III GBS infections. IVIG products that exhibit high antibody titers may be a useful therapeutic or preventive measure for infants. Further studies are needed to evaluate additional serotypes and age groups.
Antibodies* ; Complement System Proteins ; Homicide ; Humans ; Immunoglobulins* ; Immunoglobulins, Intravenous ; Infant* ; Meningitis ; Mortality ; Opsonin Proteins ; Phagocytes ; Sepsis ; Serogroup* ; Streptococcus agalactiae ; Streptococcus*

PURPOSE: Streptococcus pneumoniae is an important pathogen in children as well as in elderly people. The aim of this study was to assess the opsonization activity of pneumococal capsular antibody to seven serotypes in 23-valent pneumococcal polysaccharide vaccine before and after immunization in Korean children. METHODS: Nine children from 24 to 49 months of age were immunized with 23-valent polysaccha ride pneumococcal vaccine in Ewha Woman's University Tongdaemoon Hospital. From them, sera were collected before immunization as well as 1 month after immunization. Double-serotype opsono phagocytic killing assay was performed against seven serotypes(4, 6B, 9V, 14, 18C, 19F and 23F) of S. pneumoniae to get opsonization titer of these sera using dimethyl formamide differentiated HL-60 cells. RESULTS: After immunization, geometric mean opsonization titer of sera against serotype 18C(4,352) was the highest followed by serotype 9V(3,317), serotype 14(1,545), serotype 4(1,359). The opsonization titer against serotype 23F(342) was the lowest followed by serotype 19F(525), and serotype 6B (809). Fold increases of opsonization titer were higher against serotype 4(339.8), 9V(65.7), 19F(34.5), and 14(18.8) than those against serotype 23F(4.5), 6B(8.9), and 18C(9.9). CONCLUSION: The opsonization titers of postimmune sera against seven serotypes of S. pneumoniae were increased in many(not all) preschool children. But they showed variable values of opsonization titer in postimmune sera as well as in preimmune sera against different serotypes. The immune responses to pneumococcal polysaccharide in preschool children are not maturated sufficiently yet.
Aged ; Antibodies ; Antibody Formation* ; Child* ; Child, Preschool ; HL-60 Cells ; Homicide ; Humans ; Immunization ; Opsonin Proteins ; Pneumococcal Vaccines* ; Pneumonia ; Streptococcus pneumoniae

PURPOSE: Streptococcus pneumoniae serotype 6B and 6A are important pathogens in pneumococcal infections. It is commonly assumed that the 6B vaccines elicit antibodies cross-reacting with the 6A serotype and the cross-reactive antibodies protect against infections of 6A. To examine this assumption, we measured the opsonophagocytic capacity to serotype 6A and 6B in adults. METHODS: Twenty-four adults were immunized with pneumococcal PS vaccine that contains 6B PS. Their preimmune and postimmune sera were studied for the capacity to opsonize 6B and 6A serotypes with opsonophagocytic killing assay. RESULTS: Opsonization titers to 6B were significantly higher than those to 6A in preimmune and postimmune sera. Because significant increasesof opsonization titers were observed in adults with polysaccharide vaccines for 6A(cross-reactive) serotype as well as for 6B(vaccine) serotype, 6B PS in vaccine elicited cross-protective antibodies to 6A, but not in all cases. One adult did not have detectable levels of opsonization titers to 6A after immunization. CONCLUSION: Although 6B PS in pneumococcal PS vaccine elicits antibodies cross-reacting with 6A serotype in some adults, it may not occur always. This study should be extended to other age groups such as children and elderly people. The presence of the cross-protection should be directly determined in clinical trials of the pneumococcal vaccines as well as during the postlicensure monitoring surveys by serotyping the clinical isolates of pneumococci.
Adult ; Aged ; Antibodies ; Child ; Homicide ; Humans ; Immunization ; Opsonin Proteins ; Pneumococcal Infections ; Pneumococcal Vaccines* ; Serotyping ; Streptococcus pneumoniae ; Vaccines

BACKGROUND: Various pneumococcal vaccines have been evaluated for immunogenicity by opsonophagocytic assay (OPA). A multiplexed OPA (MOPA) for 13 pneumococcal serotypes was developed by Nahm and Burton, and expanded to 26 serotypes in 2012. The development of new conjugate vaccines with increased valence has necessitated expanded MOPAs to include these additional serotypes. In this study, we validated this expanded MOPA platform and applied to measure antibodies against 11 additional serotypes (2, 8, 9N, 10A, 11A, 12F, 15B, 17F, 20B, 22F, and 33F) in human sera. METHODS: All materials, including serum, complement, bacterial master stocks, and HL-60 cells, were evaluated for assay optimization. Following optimization, the assay was validated for accuracy, specificity, and intra- and inter-assay precision with sera from adult donors following standard protocols. The assay was applied to evaluate functional antibodies of 42 sera immunized with 23-valent pneumococcal polysaccharide vaccine (PPV23). RESULTS: The expanded MOPA platform was specific for all serotypes, with the exception of serotype 20. The assay results were highly correlated with those obtained from single-serotype OPA, indicating acceptable accuracy. The coefficients of variation were 7%–24% and 13%–39% in tests of intra- and inter-assay precision, respectively, using three quality-control samples. A MOPA that included 11 additional serotypes in the PPV23 was established and validated with respect to accuracy, specificity, and precision. The opsonic indices of immune sera were obtained using this validated assay. CONCLUSION: The expanded MOPA will be useful for evaluation of the immunogenicity of PPV23 and future conjugate vaccine formulations.
Adult ; Antibodies ; Biological Assay ; Complement System Proteins ; HL-60 Cells ; Humans ; Immune Sera ; Opsonin Proteins ; Pneumococcal Vaccines* ; Sensitivity and Specificity ; Serogroup* ; Tissue Donors ; Vaccines, Conjugate

Staphylococcus aureus is a major cause of hospital-acquired infection. Because the bacteria are very easy to become resistant to antibiotics, vaccination is a main method against S. aureus infection. Clumping factor B (ClfB) is an adhesion molecule essential for S. aureus to colonize in the host mucosa and is regarded as an important target antigen. In this study, we successfully used Escherichia coli to express a segment encoding the N1-N3 regions of ClfB protein (Truncated-ClfB) cloned from S. aureus. The protein was purified by affinity and ion exchange chromatographies and gel filtration. Rabbits were immunized three times with purified Truncated-ClfB. After that, blood was collected to prepare serum which were then used for measurement of antibody level. Phagocytosis of S. aureus opsonized by the serum was determined by a flow cytometry. Results show that the serum IgG titer reached 1:640 000. Phagocytosed S. aureus by polymorphonuclear leukocytes were significantly more when the bacteria were opsonized by the serum from Truncated-ClfB immunized rabbits than those from no immunized group (P < 0.01). Therefore, the results indicated that Truncated-ClfB could be a promising vaccine candidate against S. aureus infection.
Adhesins, Bacterial ; immunology ; Animals ; Antibodies, Bacterial ; blood ; Escherichia coli ; Flow Cytometry ; Immune Sera ; Immunoglobulin G ; blood ; Opsonin Proteins ; immunology ; Phagocytosis ; Rabbits ; Staphylococcal Infections ; immunology ; Staphylococcus aureus

Rac1 and Rac2 are essential for the control of oxidative burst catalyzed by NADPH oxidase. It was also documented that Rho is associated with the superoxide burst reaction during phagocytosis of serum- (SOZ) and IgG-opsonized zymosan particles (IOZ). In this study, we attempted to reveal the signal pathway components in the superoxide formation regulated by Rho GTPase. Tat-C3 blocked superoxide production, suggesting that RhoA is essentially involved in superoxide formation during phagocytosis of SOZ. Conversely SOZ activated both RhoA and Rac1/2. Inhibition of RhoA-activated kinase (ROCK), an important downstream effector of RhoA, by Y27632 and myosin light chain kinase (MLCK) by ML-7 abrogated superoxide production by SOZ. Extracellular signaling-regulated kinase (ERK)1/2 and p38 mitogen-activated protein kinase (MAPK) were activated during phagocytosis of SOZ, and Tat-C3 and SB203580 reduced ERK1/2 and p38 MAPK activation, suggesting that RhoA and p38 MAPK may be upstream regulators of ERK1/2. Inhibition of ERK1/2, p38 MAPK, phosphatidyl inositol 3-kinase did not block translocation of RhoA to membranes, suggesting that RhoA is upstream to these kinases. Inhibition of RhoA by Tat-C3 blocked phosphorylation of p47 PHOX. Taken together, RhoA, ROCK, p38MAPK, ERK1/2, and p47 PHOX may be subsequently activated, leading to activation of NADPH oxidase to produce superoxide.
Animals ; Cell Line ; Cell Membrane ; Cytosol ; Enzyme Inhibitors/pharmacology ; Extracellular Signal-Regulated MAP Kinases/metabolism ; Macrophage-1 Antigen/pharmacology ; Macrophages/drug effects/*metabolism/ultrastructure ; Mice ; Myosin-Light-Chain Kinase/metabolism ; Opsonin Proteins/blood/*metabolism ; *Phagocytosis ; Protein Transport ; Protein-Serine-Threonine Kinases/metabolism ; Research Support, Non-U.S. Gov't ; *Signal Transduction ; Superoxides/*metabolism ; Tetradecanoylphorbol Acetate/pharmacology ; Zymosan/*blood ; p38 Mitogen-Activated Protein Kinases/metabolism ; rhoA GTP-Binding Protein/antagonists & inhibitors/*metabolism