Background: Whole blood bactericidal assays developed by Lancefield are cumbersome to perform, not well-standardized and often lack specificity due to pre-existing naturally acquired antibodies. OPK assays using HL-60 cells as effectors and baby rabbit serum as a source of complement have recently been developed. In the present study, we have used pre-immune and immune serum samples from our recent phase 1 study of a 30-valent M protein vaccine to compare OPK activity in whole blood and HL-60 assays.
Methods: OPK assays were performed in 96-well plates in whole human blood or HL-60 cells using previously published protocols from our laboratory. OPK titers were determined using serial 2-fold dilutions of pre-immune or immune serum samples from 13 immunized subjects against M1, M3, and M12 group A streptococci. OPK titers of immune serum in whole blood or HL-60 cells were analyzed using Pearson correlations.
Results: Antibody responses in the 13 paired serum samples against M1, M3, and M12 peptides ranged from 0 to 512-fold. In general, increases in OPK titers were observed when antibody responses were >8-fold. There was significant correlation between OPK titers of immune serum samples in whole blood vs. HL-60 cells [M1 (r=.62, p=0.05), M3 (r=0.8, p=0.01), and M12 (r=0.8, p=0.01)].
Conclusions: These studies bridge results from the classic whole blood OPK assay to the newer HL-60 assay using human serum samples from vaccinated individuals. Standardized and validated HL-60 OPK assays will significantly enhance future clinical development of Strep A vaccines designed to elicit opsonic antibodies.