Chromogenic assay measuring opsonophagocytic killing capacities of antipneumococcal antisera

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 of Streptococcus 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.     

Evaluation of antibody responses to pneumococcal vaccines with ELISA and opsonophagocytic assay

Antibodies to a capsular polysaccharide (PS) provide protection against Streptococcus pneumoniae which express the homologous capsular serotype, and pneumococcal vaccines are designed to induce antibodies in the capsular PS. Levels and opsonophagocytic capacity of antibodies to the capsular PS of S. pneumoniae serotype 19F were determined by sera from adults immunized with 23-valent S. pneumoniae capsular PS vaccines. Geometric means of IgG anti-19F antibody level and specific opsonic titer rise significantly after immunization. The level of anticapsular PS antibodies for S. pneumoniae 19F serotype is fairly well correlated (r2=O.63) with the opsonophagocytic activities of sera. However, 3.7% (1/27) of serum samples display strikingly less opsonophagocytic activity than expected on the basis of their antibody level. Thus, antibody level may be of general use in predicting vaccine-induced protection among adults for 19F serotype. However, the opsonic activity data suggest that antibody levels are not always indicative of functional antibody.     

Prediction of pneumococcal conjugate vaccine effectiveness against invasive pneumococcal disease using opsonophagocytic activity and antibody concentrations determined by enzyme-linked immunosorbent assay with 22F adsorption

We compared the abilities of two serological readouts, antipolysaccharide IgG antibody concentrations and opsonophagocytic activity (OPA) titers, to predict the clinical effectiveness of the 7-valent pneumococcal conjugate vaccine (7vCRM) against invasive pneumococcal disease (IPD). We also assessed the accuracy of the previously established thresholds for GlaxoSmithKline's enzyme-linked immunosorbent assay with 22F adsorption (22F-ELISA) (≥0.2 μg/ml) and OPA assay (titer, ≥8) in predicting effectiveness. We showed that following a 3-dose 7vCRM primary vaccination, the serological response rates as determined using thresholds of ≥0.2 μg/ml IgG and an OPA titer of ≥8 corresponded well with overall effectiveness against IPD. In addition, the OPA assay seemed to better predict serotype-specific effectiveness than enzyme-linked immunoassay. Finally, when applied to post-dose-2 immune responses, both thresholds also corresponded well with the overall IPD effectiveness following a 2-dose 7vCRM primary vaccination. These results support the importance of the OPA assay in evaluating immune responses to pneumococcal conjugate vaccines.     

Respiratory-mucin inhibition of the opsonophagocytic killing of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a frequent respiratory tract colonizer in diseases in which mucociliary clearance is defective. The most striking of these is cystic fibrosis. The reasons for this organism's ability to colonize the respiratory tract and to persist there are not fully understood. Earlier studies showed that P. aeruginosa adheres preferentially to tracheobronchial mucin when compared with enterobacteria. We reasoned that if adherence to respiratory mucin protected P. aeruginosa from opsonophagocytic killing, then the ability of this organism to chronically colonize the respiratory tract could be partially explained. Using an opsonophagocytic killing assay with human polymorphonuclear leukocytes, we found that respiratory mucin protected six strains of P. aeruginosa from opsonophagocytic killing but did not protect poorly adhering strains of Escherichia coli, Staphylococcus aureus, or group B streptococci. Incubating P. aeruginosa with the mucin prior to addition to the opsonic assay inhibited phagocytic killing, whereas incubation of polymorphonuclear leukocytes with mucin did not, suggesting that inhibition was not due to an effect of mucin on leukocytes per se but was a consequence of bacterial adherence to mucin. Further studies indicated no decrease in the binding of either antibody or complement component C3 to the bacterial surface in the presence of mucin. This suggests that phagocytic inhibition may be due to a defect in uptake or destruction of mucin-coated bacteria by the leukocytes. Thus, the adherence of P. aeruginosa to respiratory mucin potentially contributes to its persistence in the respiratory tract by interfering with host immune responses.     

A flow cytometric opsonophagocytic assay for measurement of functional antibodies elicited after vaccination with the 23-valent pneumococcal polysaccharide vaccine.

Opsonophagocytosis is the primary mechanism for clearance of pneumococci from the host, and the measurement of opsonophagocytic antibodies appears to correlate with vaccine-induced protection. We developed a semiautomated flow cytometric opsonophagocytosis assay using HL-60 granulocytes as effector cells and nonviable 5, 6-carboxyfluorescein, succinimidyl ester-labeled Streptococcus pneumoniae (serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F) as bacterial targets. The flow cytometric opsonophagocytosis assay was highly reproducible (for 87% of repetitive assays the titers were within 1 dilution of the median titer) and serotype specific, with >/=97% inhibition of opsonophagocytic titer by addition of homologous serotype-specific polysaccharide. In general, opsonophagocytic titers were not significantly inhibited by the presence of either heterologous pneumococcal polysaccharide or penicillin in the serum. The flow cytometric assay could reproducibly measure functional antibody activity in prevaccination (n = 28) and postvaccination (n = 36) serum specimens from healthy adult volunteers vaccinated with the 23-valent pneumococcal polysaccharide vaccine. When compared with a standardized manual viable opsonophagocytic assay, a high correlation (r = 0.89; P 相似文献   

Specificities and opsonophagocytic activities of antibodies to pneumococcal capsular polysaccharides in sera of unimmunized young children

An enzyme immunoassay (EIA) for antibodies to pneumococcal capsular polysaccharides (Pnc PSs) detects in some cases antibodies that are cross-reactive within different Pnc PSs. Recently, it has been suggested that for detection of only serotype-specific antibodies, EIA can be modified by removing cross-reactive antibodies by absorption with an irrelevant PS, e.g., the type 22F PS. The opsonophagocytosis assay measures the functional activities of antibodies in vitro, and the results of that assay correlate with in vivo protection better than measurement of the antibody concentration by EIA. We compared these different methods for measuring antibodies to type 1, 6B, 11A, 14, 19F, and 23F Pnc PSs in the sera of unimmunized young children who had been monitored for pneumococcal carriage, acute otitis media, and acquisition of antibodies to Pnc PSs from 2 to 24 months of age. Serum samples with antibody increases after contact with a pneumococcus of a homologous serotype contained specific antibodies and often had opsonophagocytic activity (OPA) (20 of 46). In samples with antibody increases from children who had not had contact with a pneumococcus of a homologous serotype, the antibodies found to be type specific by conventional EIA were usually cross-reactive and infrequently had OPA (10 of 68). When type 22F PS absorption was used in the EIA, most of the false antibody increases were eliminated, but most of the true antibody increases were still detected and the association between the antibody concentration detected by EIA and OPA was improved. However, there were serotype-dependent differences in the frequency of OPA. Use of absorption with a heterologous PS in EIA should be encouraged, and both the specificity of EIA and the sensitivity of opsonophagocytic assays should be further evaluated and improved.     

The assay of pneumococcal capsular polysaccharide by immunodiffusion

A simple gel diffusion technique was compared with the quantitative precipitin test for evaluating concentrations of pneumococcal polysaccharide. The analysis showed that results with unknown samples compared closely by the two methods.     

Role of C5a-ase in group B streptococcal resistance to opsonophagocytic killing.

Type III group B streptococci (GBS) can be subdivided into three subtypes, RDP III-1, III-2, and III-3, on the basis of numerical analysis of HindIII restriction endonuclease digestion patterns (HindIII RDP) with their chromosomal DNAs. In the present study, the effect of C5a on opsonophagocytic killing of a representative strain from each RDP type was investigated by using a novel optical method for determining opsonophagocytic killing, and the effect of C5a-ase treatment of C5a on opsonophagocytic killing was also investigated. Pre-stimulation of polymorphonuclear leukocytes (PMNs) with C5a significantly increased opsonophagocytic killing of all three strains. The increase in killing was abolished by pretreating the C5a with GBS that express C5a-ase, a treatment that also destroyed the chemoattractant activity of the C5a. The kinetics of killing of the RDP III-2 strain differed from those of the other two strains. The survival of the RDP III-2 bacteria continued to decline over the entire 60-min incubation of the opsonophagocytic assay when PMNs were prestimulated with C5a or with C5a that had been inactivated with GBS C5a-ase (dC5a). In contrast, killing of the RDP III-1 and III-3 strains almost ceased after 20 or 60 min when PMNs were prestimulated with dC5a or C5a, respectively. A difference in bacterial killing between the III-2 strain and the III-1 and III-3 strains therefore became increasingly apparent with prolonged incubation time. The percentage of bacteria surviving in the extracellular fluid was approximately the same as the percentages of bacteria surviving in both intracellular and extracellular locations when PMNs were prestimulated with either C5a or dC5a. These data imply that the majority of bacterial killing occurred following phagocytosis and suggest that the enhanced killing of GBS following prestimulation of PMNs with C5a resulted from increased ingestion of the bacteria.     

Toll-like receptor 2 deficiency delays pneumococcal phagocytosis and impairs oxidative killing by granulocytes

Phagocytosis and killing of Streptococcus pneumoniae was compared in blood-derived wild-type (WT) and Toll-like receptor 2 (TLR2)-deficient (TLR2-/-) polymorphonuclear leukocytes (PMN). Phagocytosis of green fluorescent protein-transformed pneumococci was delayed in TLR2-/- PMN. These cells exhibited also a lower oxidative bactericidal activity against S. pneumoniae than WT PMN, suggesting that TLR2 modulates bacterial clearance in PMN.     

Fluorescent multivalent opsonophagocytic assay for measurement of functional antibodies to Streptococcus pneumoniae

We developed fluorescent mono- and multivalent opsonophagocytic assays (fOPA and fmOPA, respectively) specific for seven Streptococcus pneumoniae serotypes (4, 6B, 9V, 14, 18C, 19F, and 23F). Bacterial survival was quantitated with alamar blue, a fluorescent metabolic indicator. Both fOPA and fmOPA allow for determination of viability endpoints for up to seven serotypes with high levels of agreement to the reference method. The fmOPA eliminates colony counting, reduces serum volume, and produces results in 1 day.     

Diagnosis of pneumococcal pneumonia by enzyme-linked immunosorbent assay of antibodies to pneumococcal hemolysin (pneumolysin).

An enzyme-linked immunosorbent assay (ELISA) with a highly purified pneumolysin as the antigen was evaluated for serological diagnosis of pneumococcal pneumonia. One hundred four healthy controls were tested, and the specificity of the test was set to 95%. In samples from patients with bacteremic pneumococcal pneumonia, 82% (18 of 22) were positive, i.e., at least one serum sample had a titer above the upper normal limit or at least a twofold rise in antibody titers was noted. In nonbacteremic pneumococcal pneumonia, 45% (21 of 47) of samples were positive. All sera were negative for patients with pneumonia caused by Haemophilus influenzae, Legionella pneumophila, Chlamydia psittaci, and influenza A virus. However, in patients with a diagnosis of Mycoplasma pneumoniae infection, 8 of 25 (32%) samples were positive for antibodies to pneumolysin. All sera, including those from patients with mycoplasma infection, were negative to a protein control antigen by ELISA. Serum immunoglobulin G response to pneumolysin as measured by ELISA might thus be an aid in the laboratory diagnosis of pneumococcal pneumonia. This assay may also help to further elucidate the occurrence of dual infections with pneumococci.     

Validation of a multiplex pneumococcal serotyping assay with clinical samples

We have recently developed a rapid pneumococcal serotyping method called "multibead assay" (J. Yu et al., J. Clin. Microbiol. 43:156-162, 2005) based on a multiplexed immunoassay for capsular polysaccharides in lysates of pneumococcal cultures. The multibead assay can identify 36 serotypes (1, 2, 3, 4, 5, 6A, 6B, 7A/7F, 8, 9L/9N, 9V, 10A/10B/39/33C, 11A/11D/11F, 12A/12B/12F, 14, 15B/5C, 17F, 18C, 19A, 19F, 20, 22A/22F, 23F, and 33A/33F). More than 90% of the U.S. isolates express one of these serotypes (J. B. Robbins et al., J. Infect. Dis. 148:1136-1159, 1983). To validate the new assay, we examined 495 clinical isolates of pneumococci obtained in Brazil, Denmark, and Mexico. Pneumococci were serotyped by the Neufeld test in their countries of origin, and lysates of each strain were coded and mailed to the United States for the multibead assay at ambient temperature without any thermal protection. After breaking the code, 54 discrepancies (11% of samples) were noted, but 46 were due to nonreproducible technical problems or insufficient growth of the pneumococci. All of the isolates grew well for a second test, and therefore, the culture medium used for the multibead assay is adequate. The discrepancies persisted for eight isolates, involving the 6A, 11A, and 18C serotypes. Additional studies of the eight isolates showed that the discrepancies were due to differences in the reagents used in the multibead or Neufeld tests for these three serotypes. For instance, five isolates were typed as 6A with the Neufeld test but as nontypeable by the multibead assay. Selection of another new monoclonal antibody (Hyp6AG1) for the multibead assay resulted in all five discrepant isolates typing as 6A. This finding indicates the validity of the multibead assay and emphasizes the need to validate any new pneumococcal serotyping assay with a large number of clinical isolates from different locations. It also suggests the presence of serological subtypes among isolates expressing the 6A serotype.     

Usefulness of pneumococcal antigen detection in pleural fluid samples by immunochromatographic assay for diagnosis of pneumococcal pneumonia

This study investigated the utility of an immunochromatographic test (ICT) for the detection of Streptococcus pneumoniae antigens in pleural fluid. Antigen was detected in 15 of 19 (79%) patients with pneumococcal pneumonia. The ICT was always negative in patients with non-pneumococcal pneumonia, but was positive in three cases with a non-infectious aetiology. In patients with pneumonia for which no pathogen was identified, antigen was detected in one of 24 pleural fluids tested. The ICT can be a valuable tool for the management of pneumonia because it can detect pneumococcal antigen in pleural effusion samples.     

Multilaboratory evaluation of a viability assay for measurement of opsonophagocytic antibodies specific to the capsular polysaccharides of Streptococcus pneumoniae

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 (> or = 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.     

Performance of a pneumolysin enzyme-linked immunosorbent assay for diagnosis of pneumococcal infections

A pneumolysin-specific enzyme-linked immunosorbent assay (PLY-ELISA) for the detection of pneumolysin in urine was developed and evaluated in comparison with the commercially available Binax Now Streptococcus pneumoniae test (Binax, Portland, ME) for the diagnosis of pneumococcal infections. Assay sensitivity was evaluated using urine from 108 patients with culture-confirmed pneumococcal infections. In adults, the sensitivity and specificity of the PLY-ELISA were 56.6% and 92.2%, respectively. In children with nasopharyngeal pneumococcal carriage, PLY-ELISA and Binax Now S. pneumoniae test sensitivities were 62.5% and 87.5%, respectively, while specificities were 94.4% and 27.8%, respectively. In children with nonnasopharyngeal pneumococcal carriage, PLY-ELISA and Binax Now S. pneumoniae test sensitivities were 68.7% and 93.7%, respectively, and test specificities were 94.1% and 41.2%, respectively. The persistence of pneumolysin in urine of pneumococcal pneumonia patients decreased significantly after 4 to 6 days of treatment. Our data suggest that combining the high specificity of the PLY-ELISA with the high sensitivity of the Binax Now S. pneumoniae test would enable pneumococcal infections to be accurately diagnosed in children.     

L-ficolin and capsular polysaccharide-specific IgG in cord serum contribute synergistically to opsonophagocytic killing of serotype III and V group B streptococci

Group B streptococci (GBS; Streptococcus agalactiae) are the most common cause of neonatal sepsis and meningitis. Serotype-specific IgG antibody is known to protect neonates against GBS infections by promoting opsonophagocytosis. The L-ficolin-mediated lectin pathway of the complement is also a potential mechanism for opsonization of GBS, because L-ficolin activates the complement after binding to serotype Ib, III, V, VI, and VIII GBS. In the present study, we investigated how L-ficolin and serotype-specific IgG in cord sera contribute to opsonophagocytic killing of GBS. Neither L-ficolin nor serotype-specific IgG concentrations correlated with C3b deposition on serotype Ib and VI GBS, suggesting L-ficolin- and serotype-specific IgG-independent mechanisms of complement activation. The percentage of serotype VIII GBS killed was high regardless of the concentration of L-ficolin and IgG. In contrast, L-ficolin and serotype-specific IgG can each initiate C3b deposition on serotype III and V GBS and promote phagocytosis by polymorphonuclear leukocytes, but L-ficolin and serotype-specific IgG together promote opsonophagocytic killing to a greater extent than does either alone in vitro. This synergy was observed when serotype III-specific IgG concentrations were between 1 and 6 μg/ml and when serotype V-specific IgG concentrations were between 2 and 5 μg/ml. Concentrations of serotype III-specific IgG in cord blood above 7 μg/ml are considered protective for neonates colonized with GBS, but most neonates with IgG levels of less than 7 μg/ml do not develop GBS infections. The data presented here suggest that L-ficolin enhances opsonophagocytosis of serotype III and V GBS when serotype-specific IgG alone is suboptimal for protection.     

A new assay for the assessment of staphylococcol killing by human leucocytes

A new method is described for investigating the killing of Staphylococcus aureus by human phagocytic cells. The radioassay is based on the principle that only viable bacteria synthesize DNA and incorporate [³H]thymidine. Phagocytes are incubated with bacteria and then disrupted by a single freeze-thaw cycle. The uptake of tritiated thymidine by the remaining organisms is a measure of the killing ability of the phagocytes. The technique is simple, sensitive, rapid and requires small volumes of blood. It can be semiautomated and uses equipment readily available in an immunology laboratory and is therefore suitable for routine investigations of leucocyte function. It is likely that the technique could form the basis for measuring kill by phagocytes of any rapidly dividing organism. A time course and a normal range have been evaluated for the bactericidal capacity of 26 normal individuals.     

An analytical model applied to a multicenter pneumococcal enzyme-linked immunosorbent assay study

Pneumococcal conjugate vaccines will eventually be licensed after favorable results from phase III efficacy trials. After licensure of a conjugate vaccine for invasive pneumococcal disease in infants, new conjugate vaccines will likely be licensed primarily on the basis of immunogenicity data rather than clinical efficacy. Analytical methods must therefore be developed, evaluated, and validated to compare immunogenicity results accurately within and between laboratories for different vaccines. At present no analytical technique is uniformly accepted and used in vaccine evaluation studies to determine the acceptable level of agreement between a laboratory result and the assigned value for a given serum sample. This multicenter study describes the magnitude of agreement among 12 laboratories quantifying an identical series of 48 pneumococcal serum specimens from 24 individuals (quality-control sera) by a consensus immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) developed for this study. After provisional or trial antibody concentrations were assigned to the quality-control serum samples for this study, four methods for comparison of a series of laboratory-determined values with the assigned concentrations were evaluated. The percent error between assigned values and laboratory-determined concentrations proved to be the most informative of the four methods. We present guidelines that a laboratory may follow to analyze a series of quality-control sera to determine if it can reproduce the assigned antibody concentrations within an acceptable level of tolerance. While this study focused on a pneumococcal IgG ELISA, the methods that we describe are easily generalizable to other immunological assays.     

Biophotonic cytotoxicity assay for high-throughput screening of cytolytic killing

We have developed a highly sensitive biophotonic luciferase assay as an alternative to (51)Cr-release for assessment of cell-mediated cytotoxicity. The luciferin/ATP-dependent luminescent signal of target cells stably or transiently transfected with a firefly luciferase reporter gene (fLuc:Zeo) linearly correlates with viable target cell number. Upon incubation of fLuc:Zeo(+) target cells with CD8(+) CTLs, a rapid decrease in bioluminescence was detected that correlated with antigen-specific target cell lysis. The levels of specific lysis measured by (51)Cr-release assays correlated with the attenuation in biophotonic target cell signal, thus validating this approach as a sensitive and accurate method for the measurement of cytolysis. We show that this luminescent-based cytolytic assay (LCA) is amenable for high-throughput screening of effector cell cytolytic activity, allows for the rate of cytolysis to be measured in a single micro-plate, and permits the multiplexing of cytolytic killing with other lymphocyte functional assays such as cytokine release. Importantly, this method accurately measures the cytolytic killing of target cells that are either stably or transiently transfected with a fLuc reporter gene, and thus is ideal for monitoring cytolysis of both primary autologous and immortalized target cell lines. The versatility of the non-radioactive, high-throughput, biophotonic cytolytic assay should make this method an attractive alternative to chromium-release for quantifying effector cell cytolytic activity.