A new flow-cytometry-based opsonophagocytosis assay for the rapid measurement of functional antibody levels against Group B Streptococcus

Abstract

Opsonophagocytosis is the primary mechanism for the clearance of Group B Streptococcus (GBS) by the host, and levels of opsonic antibodies may correlate with protection in preclinical models. A killing-based opsonophagocytosis assay (OPA), can be used to determine the functional activity of vaccine-induced GBS-specific antibodies. The assay, which measures the number of bacterial colonies surviving phagocytic killing in the presence of specific antibodies and complement, is rather expensive, time-consuming and poorly standardized. Here we describe a rapid, sensitive and reproducible fluorescent OPA assay (fOPA) based on flow cytometry analysis (FACS), which allows internalized bacteria to be distinguished from those associated to the plasma membrane of phagocytic cells. Fixed GBS were labeled with pHrodo™, a fluorescent dye which dramatically increases the emitted fluorescence at the acidic conditions present in the phagocytic endosomal compartment. Labeled bacteria were incubated with HL-60 cells differentiated to phagocytes, antibodies and complement, and then analyzed by FACS. A further improvement to our method, allowing to reduce assay variability, consisted on a step of selection of effector cells among the HL-60 population. Analysis of sera from mice immunized with different GBS vaccines revealed comparable sensitivity and specificity with the traditional killing OPA assay (kOPA), and a good correlation between the fluorescent signal of bacteria internalized by HL-60 phagocytes and killing. Remarkably, the pHrodo-based approach reduced the variability observed with other fOPA assays. The obtained data indicate the proposed fOPA as a reliable and useful tool for functional antibody assessment.

Phagocytosis of neonatal pathogens by peripheral blood neutrophils and monocytes from newborn preterm and term infants

“…These inconsistent findings are likely to reflect methodological differences, as sample type (e.g., purified cells or whole blood), preparations and species of bacteria, together with the use of humoral factors (e.g., complement) vary considerably between studies (11). Although previous studies have utilized pHrodo as a tool for assessing phagocytosis, none have extended its use to simultaneous analysis of multiple cell types, or to whole blood assays (18,21). We found that preterm infants possess a decreased proportion of whole-blood phagocytes capable of phagocytosing the common neonatal pathogens SE, EC and, to a lesser extent, SA compared with term infants.…”

An improved flow cytometry assay to monitor phagosome acidification

“…In contrast to latex bead fluorescence, conjugation of pHrodo TM probe with particles (bacteria or dead cells for example) is a reliable method to monitor phagosome acidification following engulfment (Miksa et al, 2009;Fabbrini et al, 2012;Toda et al, 2012;Neaga et al, 2013). However, this method is poorly informative in terms of particle association with cells, engulfment efficiency within associated particles and acidification rates.…”

“…This led to the development of fluorescence-based methods to monitor phagocytosis and acidification using pHrodo-labelled particles that have an increased fluorescence in acidic compartments (Miksa et al, 2009;Fabbrini et al, 2012;Toda et al, 2012;Neaga et al, 2013;Tan et al, 2013). However, the use of biodegradable particles such as bacteria or dead cells does not allow precise monitoring of the efficiency of particle association and engulfment by cells.…”

Distinct Cellular Pathways for Induction of CD4+ T Cell–Dependent Antibody Responses to Antigen Expressed by Intact Bacteria Versus Isolated Soluble Antigen

“…Alum (Alhydrogel 2%) was obtained from Brenntag Biosector. A stimulatory 30 mer CpG-containing oligodeoxynucleotide (CpG-ODN) was synthesized as previously described (30 Labeling R614-OVA 323-339 and OVA with pHrodo R614-OVA 323-339 was labeled as described earlier (31). In brief, bacteria were grown in Todd Hewitt broth (BD Biosciences) and heat killed, as mentioned above.…”