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. 2023 Nov 9;11(11):1703.
doi: 10.3390/vaccines11111703.

Development of A Standardized Opsonophagocytosis Killing Assay for Group B Streptococcus and Assessment in an Interlaboratory Study

Stephanie Leung  1 , Clare F Collett  1 , Lauren Allen  1 , Suzanna Lim  2 , Pete Maniatis  3 , Shanna J Bolcen  3 , Bailey Alston  4 , Palak Y Patel  3 , Gaurav Kwatra  5   6 , Tom Hall  2 , Stephen Thomas  1 , Stephen Taylor  1 , Kirsty Le Doare  2 , Andrew Gorringe  1
Affiliations

Development of A Standardized Opsonophagocytosis Killing Assay for Group B Streptococcus and Assessment in an Interlaboratory Study

Stephanie Leung et al. Vaccines (Basel). .

Abstract

The placental transfer of antibodies that mediate bacterial clearance via phagocytes is likely important for protection against invasive group B Streptococcus (GBS) disease. A robust functional assay is essential to determine the immune correlates of protection and assist vaccine development. Using standard reagents, we developed and optimized an opsonophagocytic killing assay (OPKA) where dilutions of test sera were incubated with bacteria, baby rabbit complement (BRC) and differentiated HL60 cells (dHL60) for 30 min. Following overnight incubation, the surviving bacteria were enumerated and the % bacterial survival was calculated relative to serum-negative controls. A reciprocal 50% killing titer was then assigned. The minimal concentrations of anti-capsular polysaccharide (CPS) IgG required for 50% killing were 1.65-3.70 ng/mL (depending on serotype). Inhibition of killing was observed using sera absorbed with homologous CPS but not heterologous CPS, indicating specificity for anti-CPS IgG. The assay performance was examined in an interlaboratory study using residual sera from CPS-conjugate vaccine trials with international partners in the Group B Streptococcus Assay STandardisatiON (GASTON) Consortium. Strong correlations of reported titers between laboratories were observed: ST-Ia r = 0.88, ST-Ib r = 0.91, ST-II r = 0.91, ST-III r = 0.90 and ST-V r = 0.94. The OPKA is an easily transferable assay with accessible standard reagents and will be a valuable tool to assess GBS-specific antibodies in natural immunity and vaccine studies.

Keywords: Group B Streptococcus; OPKA; correlates of protection; neonatal antibodies; opsonophagocytosis assay; transplacental antibodies; vaccines.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Figures

Figure 1
Figure 1
Binding of serotype-specific rabbit antisera (SSI Diagnostica, Hillerød, Denmark) to 49 GBS isolates representing serotypes Ia, Ib, II, III and V was determined by flow cytometry. Each strain was evaluated against both homologous and heterologous typing antisera in duplicate (mean fluorescence values and standard deviations are listed in Table S1 of Supplementary Materials).
Figure 2
Figure 2
Performance of 49 GBS isolates in OPKA using serotype-matched standard human reference sera (SHRS). The selected isolate is shown in bold. Experiments were carried out in duplicate with plotted mean values.
Figure 3
Figure 3
During assay optimization, several parameters were investigated including (A) incubation temperature, (B) incubation time, (C) complement concentration and (D) use of individual or pooled batches of complement. Experiments were carried out in duplicate with geometric mean and standard deviation shown.
Figure 4
Figure 4
Schematic of optimized opsonophagocytic killing assay (OPKA). The assay comprises two stages: test serum (serially diluted) is incubated with bacteria to allow for opsonization to occur; followed by addition of baby rabbit complement and dHL60 cells to initiate phagocytic killing. COH agar plates are inoculated in the final step by plating 10 µL of assay mixture and tilting to form streaks, followed by an overnight incubation. Colonies (highlighted green by image analysis software) are enumerated using manual or digital image analysis methods. Killing titers are calculated as the 50% reciprocal dilution point compared to the average CFU in serum-free controls.
Figure 5
Figure 5
Homologous and heterologous CPS mixes were prepared (1 in 5 dilution series) and pre-incubated with SHRS at a known killing titer. The corresponding GBS serotype isolate was added and the remainder of the assay was carried out. Inhibition experiments were carried out in triplicate with mean and standard deviation shown.
Figure 6
Figure 6
Determination of anti-CPS IgG concentration required to mediate 50% killing. Anti-capsular polysaccharide antibody concentrations denoted on the x-axis were calculated corresponding to the starting dilution of SHRS used for the OPKA. Experiments were carried out in duplicate with geometric mean and geometric standard deviation shown.
Figure 7
Figure 7
Performance of OPKA assessed by interlaboratory study. Four sites across three continents participated and tested the same proficiency serum panel with multiple operators. (A) Distribution of OPKA titers obtained are shown for each laboratory and serotype. Samples were tested in duplicate with geometric mean values plotted. The solid black line represents the population median and the dashed black line represent 25% and 75% percentiles. (B) Pairwise comparisons between laboratories by Pearson’s analysis demonstrated strong correlations, as detailed in Table 2.
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