Measuring immune responses to pneumococcal vaccines

Abstract

Quantitative assays that measure immune response to pneumococcal vaccines are not only important for the evaluation of vaccine immunogenicity and efficacy, but are also utilized in the clinical diagnosis of immune deficiency syndromes. Analytical methods have progressed in order to meet changing demands in both of these areas, from early methods to ELISA, and most recently multiplex bead array assays and opsonophagocytosis assays (OPA). It is necessary to understand the evolution of such techniques and the criteria for their interpretation in order to better inform the application of currently available methods, and to guide future investigation into assay development.

Keywords: Immune deficiency; Immune response; Pneumococcal; Streptococcus pneumoniae; Vaccine.

Figure 1.. WHO ELISA for measurement of pneumococcal IgG antibodies.

The figure represents a typical plate well in WHO ELISA. The left side of the figure depicts the initial pre-absorption of sera with cell wall polysaccharide (C-PS) and 22F polysaccharide to neutralize non-protective antibodies (shown in brown). The right side of the figure illustrates binding of serum pneumococcal IgG antibodies (shown in green) to serotype-specific pneumococcal capsular polysaccharide. Anti-human IgG antibodies (shown in blue) are bound to serum IgG antibodies. Using a chromogenic substrate, optical density is measured, and then converted to antibody concentration.

Figure 2.. Classical opsonophagocytosis assay to measure killing of pneumococci.

The left side of the figure depicts a plate well used in classical OPA, in which serum is added to a mixture of phagocytes, complement, and pneumococcus of a given serotype. Dilutions of this mixture are plated on a standard agar plate (center). Bacterial colonies are counted to generate a killing curve. On the far right a representative example of a killing curve is shown. For each of the serum dilutions indicated (X axis), the average surviving colony-forming units (CFU) is shown on the y axis. The bold, solid horizontal line indicates the maximum CFU (i.e., 0% killing), and the dashed horizontal line indicates the CFU with 50% killing.

Figure 3.. Multiplex opsonophagocytosis assay (MOPA) to measure killing of multiple pneumococcal serotypes.

On the left a typical MOPA plate well is shown, in which serum is added to a mixture of phagocytes (differentiated HL-60 cells), rabbit complement, and multiple serotypes of pneumococci, each carrying resistance to a different antibiotic. Dilutions of this mixture are plated on antibiotic-containing media (center) to select for growth of a single serotype. Bacterial colonies are enumerated using an automated counter to generate a killing curve as shown on the right (see Figure 2 for a detailed description).