Multilaboratory Evaluation of a Viability Assay for Measurement of Opsonophagocytic Antibodies Specific to the Capsular Polysaccharides of Streptococcus pneumoniae

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.Vaccine-induced protection to Streptococcus pneumoniae (pneumococcus) has been determined through vaccine efficacy trials for both polysaccharide (Ps) vaccines (1, 4, 22) and Psprotein conjugate vaccines (2,5,8). Trials of these pneumococcal (Pnc) vaccine formulations have shown various efficacies for protection depending on the end point being measured and the population being studied. These trials are costly and difficult to perform given the large sample size. In addition, pneumococcus has 90 different capsular serotypes, with the majority of disease being caused by about 30 of these 90 serotypes. Distribution of these serotypes also varies with the geographical region, making the estimation of the burden of disease and the impact of vaccination rather difficult (3, 9, 10).Efforts have been made for the identification and standardization of laboratory correlates of protection that can aid vaccine efficacy trials in the estimation of vaccine-induced protection. Currently, a highly standardized enzyme-linked immunosorbent assay (ELISA) is available (www.vaccine.uab.edu) for the evaluation of infant sera. Several modifications to the protocol described by Quartaert et al. (20) allowed for the measurement of Ps-specific antibodies in children and adults (6,19,18). Adults can have cross-reactive antibodies, which confound the measurements of immunoglobulin G (IgG) antibodies by ELISA, especially if absorption with a nonrelevant serotype is not performed prior to testing (6,7,26). These c...

Use of HL-60 Cell Line To Measure Opsonic Capacity of Pneumococcal Antibodies

“…In contrast, several experienced European laboratories in various countries reported difficulties in achieving reproducible differentiation of HL-60 cells into granulocytes with DMF. Although some European laboratories were successful in using HL-60 (72), these diverse international experiences became a concern in adopting the HL-60 derived granulocytes as the effector cells for pneumococcal OPAs.…”

“…Maintenance of HL-60 is possible in RPMI 1640 supplemented with 2 mM L-Glutamine and 10 to 20% heat-inactivated FBS from various sources (HyClone, Logan, Utah; JRH Biosciences, Lenexa, Kans.) (29,72). Under these conditions, HL-60 cultures can be maintained by diluting the cells with a fresh medium to a density of 10 5 viable cells/ml when the cell density reaches 10 6 cells/ml.…”

“…This method mimics the opsonophagocytic process occurring in vivo and has been used for many years. Recently, an evaluation of this methodology indicated that this assay has a high degree of reproducibility across multiple laboratories (72).…”

“…Therefore, for a standardized bioassay it is critical to standardize and optimize differentiation conditions to provide reproducible yields of granulocytes suitable for use as effector cells within the OPA. Optimized conditions have been described by various authors for differentiation into neutrophils and these include 1.25% (vol/vol) of DMSO in a period of 5 to 7 days (10,19), 100 mM DMF in a period of 5 days (57,72,73), or 0.1 M all-trans-retinoic acid (ATRA) in a period of 5 days (11). In some cases, comparisons between inducers indicate they can be synergistic and are more effective when they are used in combination (11,13).…”

Antibodies Specific for the High-Molecular-Weight Adhesion Proteins of NontypeableHaemophilus influenzaeAre Opsonophagocytic for both Homologous and Heterologous Strains

“…In previous work, we characterized human opsonophagocytic antibodies recognizing nontypeable Haemophilus influenzae by using an assay modified from one developed for the measurement of antibodies against Streptococcus pneumoniae (51,52). In that earlier work, we demonstrated that antibodies directed against the HMW1/ HMW2-like proteins were major contributors to the opsonophagocytic activity mediated by naturally acquired human antibodies (65).…”