Flow cytometry-based phagocytosis assay for sensitive detection of opsonic activity of pneumococcal capsular polysaccharide antibodies in human sera

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Abstract

The development of efficient vaccines against Streptococcus pneumoniae is of major importance for public health. The efficacy of pneumococcal vaccination and induced protection are thought to be reflected by the opsonic antibody titers in sera from vaccines. We describe a novel two-color flow cytometry technique for quantification of antibody-mediated pneumococcal phagocytosis. Serum-opsonised fluorescein-isothiocyanate (FITC)-labelled S. pneumoniae were allowed to attach to neutrophils, split into two aliquots and further incubated either at 4°C (to avoid phagocytosis) or 37°C (to allow phagocytosis). Cell-surface residual opsonic IgG was detected by phycoerythrin (PE)-conjugated anti-human IgG in both samples. The fraction of FITC-labelled bacteria phagocytosed via antibody (Fi) could be estimated from FITC and PE labels, and reflected the opsonic activity of sera. The technique displayed high sensitivity for the detection of opsonic antibodies, as shown by experiments using pre- and post-immune sera, which documented significantly increased phagocytosis after vaccination, and the observed increase in phagocytosis rates at higher antibody levels. The intrinsic variation of the assay was low, and could be further reduced by the use of effector cells from donors with similar IgG receptor (FcγR) allotypes. The method described in this study should be generally applicable to test vaccine efficacy, to evaluate the interaction of bacteria and phagocytes, and to discriminate between antibody-mediated and antibody-independent interactions between bacteria and phagocytes.

Introduction

Host defence against encapsulated bacteria, such as Streptococcus pneumoniae, depends on the presence of opsonic antibodies specific for capsular polysaccharide (PS) (Musher et al., 1986). Vaccination strategies therefore aim to elicit antibodies with opsonic activity against pneumococci. Antibody titers as measured by ELISA may not adequately reflect the presence of functional antibodies, capable of triggering leukocyte effector functions. Phagocytosis of bacteria triggered by the sera of vaccinees is thought to be a superior read-out, in that it constitutes a more reliable parameter for evaluation of the clinical protective efficacy of vaccines Giebink et al., 1980, Musher et al., 1986, Viðarsson et al., 1994.

Phagocytosis is initiated by the binding of bacteria to phagocytes, a process facilitated by the interaction of specific antibody with Fc receptors (FcR) on leukocyte surfaces. Engagement of FcR initiates signaling cascades and may finally result in the engulfment of bacteria by the phagocyte and the formation of a phagosome, which is required for efficient anti-bacterial activity Griffin et al., 1975, Van der Pol and Van de Winkel, 1998, Viðarsson and Van de Winkel, 1998.

A number of phagocytosis assays have been described that could be used for vaccine evaluation. Microscopy-based assays, including conventional light- and fluorescent microscopy Van den Dobbelsteen et al., 1991, Gordon et al., 2000 as well as advanced confocal laser scan microscopy techniques Sanders et al., 1995, Weaver et al., 1996 are qualitatively reliable techniques, but quantification of phagocytosis is laborious, which limits its suitability for testing large numbers of sera. Indirect techniques to assess phagocytosis rates may be more rapid, but have other limitations. Plating techniques have notoriously high degrees of variation. Phagocytosis techniques using radioactively labelled bacteria require specialised laboratories and personnel for reasons of safety. In addition, some of these techniques were reported to depend on the presence of complement Viðarsson et al., 1994, Romero-Steiner et al., 1997, and may therefore not directly reflect the opsonic qualities of PS-specific antibodies.

Flow cytometer (FACS)-based phagocytosis assays have been proposed as attractive and rapid alternatives to the above Sveum et al., 1986, Sanders et al., 1995, Obaro et al., 1996, Jansen et al., 1998, Martinez et al., 1999. In these FACS-assays, phagocytes are generally incubated at 37°C with fluorescent bacteria pre-opsonised with an appropriate dilution of serum. Read-out systems that are thought to reflect bacterial phagocytosis include the mean fluorescence of effector cells and/or the percentages of fluorochrome positive leukocytes, or serum-titers at which a defined read-out value is detected. Although these techniques are rapid and efficient for large scale testing of sera, they possess intrinsic drawbacks, such as quenching of fluorescence intensity upon internalisation, an inability to distinguish between adherent and internalised bacteria in most cases, or a failure to determine antibody-mediated phagocytosis.

We developed a new FACS-based method that permits distinction between adherent and phagocytosed material and the estimation of antibody-mediated bacterial phagocytosis. This assay proved to be highly sensitive and showed limited inter- and intra-experiment variation.

Section snippets

Human sera

Sera from seven children (aged 0.5–9 years old) immunized with the 7-valent pneumococcal conjugate vaccine (Lederle Wyeth, Pearl River, NY) and seven adults immunized with the 23-valent pneumococcal polysaccharide vaccine (Pneumovax®, Merck, Sharp and Dohme, Haarlem, The Netherlands) were collected before and 1 month after immunisation. All sera used in this study were collected at the University Medical Center Utrecht (UMCU), The Netherlands, and are representative of a larger panel of tested

Fluorescent labelling and opsonisation of pneumococci

A single batch of FITC-labelled bacteria was used in all experiments. The labelling proved stable for at least 3 years in storage, as demonstrated by one discrete peak (Fig. 1A) and identical fluorescence intensity over time by FACS analysis.

Serum-opsonised bacteria stained with PE-conjugated goat F(ab′)2 anti-human IgG antiserum showed both green and red fluorescence in FACS (Fig. 1B). When examined with confocal fluorescence microscopy, bacteria showed a central green fluorescence surrounded

Discussion

Host protection against invasive pneumococcal disease depends crucially on the presence of opsonic anti-capsular polysaccharide antibodies. Therefore, the evaluation of efficacy of pneumococcal polysaccharide and conjugate vaccines involves the determination of anti-PS antibody responses. Although measurements of polysaccharide-specific antibody concentration by ELISA have been used extensively Käyhty et al., 1995, Åhman et al., 1996, Dagan et al., 1997, ELISA titers do not correlate well with

Acknowledgements

The authors thank Drs. G. Rijkers and L. Sanders for kindly providing sera from patients. This study was supported by the Netherlands Organisation for Scientific Research (NWO grant no. 95-10-624), and the Argentinean Research Council (CONICET grant no. 1377).

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