A novel flow cytometry-based assay for the quantification of antibody-dependent pneumococcal agglutination

RESEARCH ARTICLE A novel flow cytometry-based assay for the quantification of antibody-dependent pneumococcal agglutination Marrit N. Habets☯, Saskia van Selm*☯, Christa E. van der Gaast—de Jongh, Dimitri A. Diavatopoulos, Marien I. de Jonge Laboratory of Pediatric Infectious Diseases, Department of Pediatrics, Radboud Institute for Molecular Life Sciences, Radboud university medical center, Nijmegen, The Netherlands a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Habets MN, van Selm S, van der Gaast— de Jongh CE, Diavatopoulos DA, de Jonge MI (2017) A novel flow cytometry-based assay for the quantification of antibody-dependent pneumococcal agglutination. PLoS ONE 12(3): e0170884. https://doi.org/10.1371/journal. pone.0170884 Editor: Paulo Lee Ho, Instituto Butantan, BRAZIL Received: March 1, 2016 Accepted: January 12, 2017 Published: March 13, 2017 Copyright: © 2017 Habets et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper. Funding: This study was funded by the EU (Eurostars, Pneumonav project, grant number: 9285, https://www.eurostars-eureka.eu/project/id/ 9285). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. ☯ These authors contributed equally to this work. * Abstract The respiratory pathogen Streptococcus pneumoniae is a major cause of diseases such as otitis media, pneumonia, sepsis and meningitis. The first step towards infection is colonization of the nasopharynx. Recently, it was shown that agglutinating antibodies play an important role in the prevention of mucosal colonization with S. pneumoniae. Here, we present a novel method to quantify antibody-dependent pneumococcal agglutination in a high-throughput manner using flow cytometry. We found that the concentration of agglutinating antibodies against pneumococcal capsule are directly correlated with changes in the size and complexity of bacterial aggregates, as measured by flow cytometry and confirmed by light microscopy. Using the increase in size, we determined the agglutination index. The cutoff value was set by measuring a series of non-agglutinating antibodies. With this method, we show that not only anti-polysaccharide capsule antibodies are able to induce agglutination but that also antiPspA protein antibodies have agglutinating capabilities. In conclusion, we have described and validated a novel method to quantify pneumococcal agglutination, which can be used to screen sera from murine or human vaccination studies, in a high-throughput manner. Introduction Nasopharyngeal colonization with the respiratory pathogen Streptococcus pneumoniae is a prerequisite for the development of pneumococcal disease. Following dissemination of bacteria to the ear, lung, bloodstream or brain, otitis media, pneumonia, sepsis or meningitis may develop, respectively. Several mucosal defense mechanisms, such as antibody-mediated opsonization and opsonophagocytosis by phagocytes, have been suggested to be important in the reduction or complete prevention of colonization [1,2]. Recently, Roche et al. (2015) showed that the presence of agglutinating antibodies on the mucosal surface plays an important role in the prevention of pneumococcal colonization in a mouse model of colonization and transmission [3]. The agglutinating properties of antibodies raised against novel vaccine candidates PLOS ONE | https://doi.org/10.1371/journal.pone.0170884 March 13, 2017 1 / 11 A novel assay for the quantification of pneumococcal agglutination might therefore be predictive for efficacy, and would be an important parameter to include in clinical trials. The agglutinating properties of antibodies against S. pneumoniae have long been known. In 1902 Neufeld described agglutination of pneumococci with specific antisera, visible as capsular swelling and clumping of bacteria, which became known as the quellung reaction [4]. Classification of pneumococci by specific serological reactions was described in 1913 [5,6], and the identification of new serotypes followed over the years, leading to the description of 80 distinct serotypes in 1960 [7]. Since then, the quellung reaction using polyclonal rabbit antisera has been the ‘gold standard’ for serotyping. Capsular swelling, which is usually accompanied by agglutination, is typically visualized microscopically. Latex agglutination or slide agglutination is viewed macroscopically. [8,9]. While the determination of an agglutination titer for serum against several bacteria has been described using a tube or well agglutination test, where a titer is determined using serially diluted serum that is mixed with a constant quantity of bacteria [10], variability in individual interpretation of results makes it difficult to standardize this method. In addition, this method is relatively time consuming and therefore not very suitable for high-throughput use. Mucosal protection against colonization by agglutinating capsule-specific antibodies is thought to be mediated predominately by immunoglobulin G. Although IgA1 antibodies, which represent the most abundant immunoglobulin subclass present on the airway mucosa, can also induce bacterial aggregates, the expression of IgA1 protease by S. pneumoniae has been shown to negate this effect [3]. The pneumococcal conjugate vaccines (PCVs) induce high amounts of systemic IgG against several different types of capsular polysaccharides. Due to active transport to the mucosal surface via the neonatal Fc receptor, these antibodies also provide protection against pneumococcal colonization [11,12]. Recent studies have shown that agglutination by anti-pneumococcal IgG antibodies contributes to protection against pneumococcal colonization [3,13]. Since the agglutinating effect of antibodies has shown to be an important factor in the protection against pneumococcal colonization, there is a clear need for adequate methods to assess and quantify this antibody functionality. However, to date, there is no standardized method to measure pneumococcal agglutination. Here, we developed a high-throughput method to screen serum samples for their agglutinating potential of various pneumococcal strains, using flow cytometry. Using this novel method, we assessed the agglutinating potential of both capsule-specific antibodies and antibodies generated against the pneumococcal surface protein A (PspA). Materials and methods Pneumococcal strains The Streptococcus pneumoniae serotype 4 strain TIGR4 [14] and the serotype 19F strain EF3030 [15] were used in agglutination experiments with anti-capsular antibodies. Non-encapsulated strains (...truncated)