Development of an Opsonophagocytic Killing Assay Using HL-60 Cells for Detection of Functional Antibodies against Streptococcus pyogenes

The clinical development of group A streptococcal (GAS) vaccines will require the implementation of a standardized, high-throughput assay to measure the activity of functional opsonic antibodies in vaccine recipients. In the present study, we adapted and modified the HL-60-based protocol that was developed for the detection of opsonic antibodies against Streptococcus pneumoniae for use with multiple M types of GAS. Modifications of the assay conditions permitted the evaluation of 21 different M types of GAS in the assay. The specificity of the antibody-mediated opsonization was demonstrated by inhibition with homologous, but not heterologous, M proteins. Maximum rates of opsonophagocytic killing (OPK) of 14 different M types promoted by rabbit antiserum against the 30-valent M protein-based vaccine were comparable in whole-blood and HL-60 assays. Data are also presented showing OPK serum titers (opsonic index) of naturally acquired human antibodies present in IVIG [intravenous immune globulin (human)]. Results of the HL-60 assay performed on different days using 21 different M types of GAS and IVIG as the antibody source were significantly concordant. This report indicates that the OPK assay conditions may be optimized for the measurement of opsonic antibodies against a number of epidemiologically important M types of GAS and, once standardized, should facilitate the clinical development of effective vaccines to prevent these infections.IMPORTANCE Measuring functional opsonic antibodies against group A streptococci is an important component of the clinical development path for effective vaccines. Prior studies have used an assay developed over 60 years ago that relied on whole human blood as the source of phagocytes and complement, both of which are critical components of antibody-mediated killing assays. In this study, we adapted an assay that uses the HL-60 human promyelocytic leukemia cell line as phagocytic cells and baby rabbit serum as a source of complement for detection of opsonic antibodies against group A streptococci. On the basis of some of the known biological characteristics of the bacteria, we modified the assay conditions to support the evaluation of 21 epidemiologically important M types and demonstrated the utility and reproducibility of the assay for measurement of functional opsonic antibody levels.