Use of highly encapsulated Streptococcus pneumoniae strains in a flow-cytometric assay for assessment of the phagocytic capacity of serotype-specific antibodies

Abstract

A phagocytosis assay for Streptococcus pneumoniae based on flow cytometry (FACS) with human polymorphonuclear cells and human complement was developed for the study of human vaccination antisera. Human prevaccination sera already contain high levels of C-polysaccharide (C-PS) antibodies, which are not protective in humans but which might give false positive results in a flow-cytometry-based assay. Cultures of S. pneumoniae grown to log phase on three consecutive days, followed by heat inactivation, yielded stable and highly encapsulated strains for serotypes 6A, 6B, 14, 19F, and 23F. As a result, only serotype-specific antibodies were able to facilitate phagocytosis of these strains, whereas no phagocytosis was observed with antibodies against C-PS or pneumococcal surface proteins. No, or weak, phagocytosis was observed with human prevaccination sera, whereas in general, postvaccination antisera facilitated phagocytosis. A highly significant correlation was observed between enzyme-linked immunosorbent assay titers and FACS phagocytosis titers (r = 0.98, P < 0.001) for serotype 23F pneumococci with human vaccination antisera. For all serotypes, interassay variation was below 10%. Major advantages of this assay over the classical killing assay are that (i) limited amounts of sera are required (10 microliter per titration curve), (ii) 600 samples can be processed in one day by one person, and (iii) cells can be fixed and measurement of the samples can be performed up to 1 week later.

FIG. 1

(a) PMN cell gating (R1) based on their specific forward (FSC) and right angle (SSC) light scatter. (b) Analysis of phagocytosis of fluorescence-labeled S. pneumoniae by PMN cells. The histograms show FITC-positive and -negative PMN cells (discriminated by marker M1) for different serum concentrations: 10% (A), 2% (B), 0.4% (C). Negative controls: PMN cells without serum (D), PMN cells without serum and complement (E), PMN cells only (F).

FIG. 2

Effect of growth phase and C-PS preincubation on anti-C-PS antibody-mediated phagocytosis. A serotype 14 strain was grown to the stationary phase (panels A and C) or the log phase (panels B and D) and tested with an antiserum against C-PS and serotype 14 pneumococci. (A) Phagocytosis of the stationary-phase strain with an antiserum against C-PS (■) and serotype-specific antiserum (▴). (B) Phagocytosis of log-phase strain with an antiserum against C-PS (■) and serotype-specific antiserum (▴). (C) Preincubation of a C-polysaccharide antiserum with C-polysaccharide before opsonization of a stationary strain. C-polysaccharide antiserum was used alone (▵) or preincubated with C-PS (□). (D) Influence of C-PS preincubation and the use of the log-phase strain on C-PS antibody-mediated phagocytosis. C-polysaccharide antiserum was used alone (▵) or preincubated with C-PS (□).

FIG. 3

Anti-C-PS antibodies do not promote phagocytosis of highly encapsulated strains. A serotype 19F strain was tested with an antiserum against C-PS (■) and 19F pneumococci (▴).

FIG. 4

Comparison of the effect of three inactivation methods on phagocytosis mediated by anti-C-PS and serotype-specific antisera. Serotype 19F strains were inactivated by heat (60°C) (circles), 2% paraformaldehyde (triangles), or 70% ethanol (squares) and phagocytized by an antiserum against C-PS (open symbols) or 19F pneumococci (closed symbols).

FIG. 5

Rabbit anti-pneumococcal protein antisera do not promote phagocytosis of heat-inactivated strains grown to log phase three times. A serotype 19F strain, grown to stationary phase (closed symbols) or to log phase three times (open symbols) was tested with rabbit prevaccination (trangles) and postvaccination (squares) sera against hydrophobic protein fractions.

FIG. 6

Competitive inhibition of phagocytosis with C-polysaccharide or serotype-specific polysaccharide. Rabbit antisera were preincubated with either C-polysaccharide or serotype-specific polysaccharide, and a phagocytosis assay was performed with the highly encapsulated strains. Figure shows preincubation with C-polysaccharide (open bars) and with serotype-specific polysaccharides (hatched bars) (n = 3).

FIG. 7

Correlation between ELISA IgG titers and FACS-based phagocytosis titers for human prevaccination (○) and postvaccination (•) sera. ELISA titers and phagocytosis titers were determined for 48 pre- and postvaccination antisera.