Pneumococcal surface protein (PspA) is a virulence factor expressed by all clinical isolates of Streptococcus pneumoniae. PspAs are variable in structure and have been grouped into clades and cross-reacting families based on sequence similarities and immunologic cross-reactivity. At least 98% of PspAs are found in PspA families 1 or 2. PspA has been shown to interfere with complement deposition on pneumococci, thus reducing opsonization and clearance of bacteria by the host immune system. Prior studies using pooled human sera have shown that PspA interferes with C3 deposition on a single strain of S. pneumoniae, WU2, and that mouse antibody to PspA can enhance the deposition of C3 on WU2. The present studies have demonstrated that these previous findings are representative of most normal human sera and each of seven different strains of S. pneumoniae. It was observed that PspAs of PspA families 1 and 2 could inhibit C3 deposition in the presence of immunoglobulin present in all but 3 of 22 normal human sera. These studies have also demonstrated that rabbit and human antibody to PspA can enhance the deposition of C3 on pneumococci expressing either family 1 or 2 PspAs and either capsular types 2, 3, or 11. A vaccine candidate that can elicit immunity that neutralizes or compensates for S. pneumoniae's ability to thwart host immunity would be of value.

Introduction: Commercially available enzymelinked immunosorbent assay (ELISA) kits designed for pertussis diagnostic purposes are frequently used to assess antibody responses to pertussis vaccines in clinical trials, but have limited accuracy and are not calibrated against international standards. We developed a new electrochemiluminescence (ECL)-based multiplexed assay and compared its performance to two commercial Bordetella pertussis ELISA kits and to historical in-house ELISAs. Methods: The ECL assay quantifies serum concentrations of antibodies against four B. pertussis antigens: pertussis toxin (PT), filamentous hemagglutinin (FHA), pertactin (PRN), and fimbrial agglutinogen (FIM). The assay was validated for precision, accuracy, dilutability, lower limit of quantification, and specificity.Sera from a clinical trial (CTRI/2016/11/007434) were used to compare the ECL assay to two commercial ELISA kits available from GenWay BioTech and Demeditec Diagnostics for accuracy, linearity, specificity, and concordance to both internal (WWO-2-043) and international (NIBSC 06/140) references. Sera from four clinical trials (NCT02587520, NCT00255047, NCT00347958, NCT01346293) were used to compare the concordance to clinical ELISAs. Informed consent was ensured prior to using any sera. Results: Precision, accuracy, dilutability, lower limit of quantification, and specificity were demonstrated for the ECL assay. Concordance between the ECL assay and established clinical ELISAs was met for antibody responses to PT, FIM, and PRN, but not for FHA. The ECL assay demonstrated higher accuracy and linearity than the ELISA kits. While concordance between the ECL and commercial kits was low, the ECL assay better distinguished between preand post-vaccination clinical samples. Conclusion:The new ECL assay was validated for the quantitative evaluation of anti-PT, anti-FHA, anti-FIM, and anti-PRN IgG antibodies in samples from clinical trials, and demonstrated equivalent or better performance than two commercially available ELISA kits.

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