Development and Use of a Serum Bactericidal Assay Using Pooled Human Complement To Assess Responses to a Meningococcal Group A Conjugate Vaccine in African Toddlers

Abstract

A meningococcal group A polysaccharide (PS) conjugate vaccine (PsA-TT) has been developed for African countries affected by epidemic meningitis caused by Neisseria meningitidis. Complement-mediated serum bactericidal antibody (SBA) assays are used to assess protective immune responses to meningococcal vaccination. Human complement (hC=) was used in early studies demonstrating antibody-mediated protection against disease, but it is difficult to obtain and standardize. We developed and evaluated a method for sourcing hC= and then used the SBA assay with hC= (hSBA) to measure bactericidal responses to PsA-TT vaccination in 12-to 23-month-old African children. Sera with active complement from 100 unvaccinated blood donors were tested for intrinsic bactericidal activity, SBA titer using rabbit complement (rSBA), and anti-group A PS antibody concentration. Performance criteria and pooling strategies were examined and then verified by comparisons of three independently prepared hC= lots in two laboratories. hSBA titers of clinical trial sera were then determined using this complement sourcing method. Two different functional antibody tests were necessary for screening hC=. hSBA titers determined using three independent lots of pooled hC= were within expected assay variation among lots and between laboratories. In African toddlers, PsA-TT elicited higher hSBA titers than meningococcal polysaccharide or Hib vaccines. PsA-TT immunization or PS challenge of PsA-TTprimed subjects resulted in vigorous hSBA memory responses, and titers persisted in boosted groups for over a year. Quantifying SBA using pooled hC= is feasible and showed that PsA-TT was highly immunogenic in African toddlers.

A high-throughput, bead-based, antigen-specific assay to assess the ability of antibodies to induce complement activation

“…Here whole pathogens, components, or epitope-scaffolds can be individually coupled to beads, with simply coupling adaptions, to fully interrogate the response. Furthermore, the requirement for fresh human plasma as the gold standard in SBA reduces throughput and may introduce variation (Bash et al, 2014), even though we have shown that human complement is comparable across donors. Conversely, the high-throughput assay described here can be adapted for profiling of almost any antigen, including glycan-, lipid-, or protein-based antigens, that may be coupled to a bead in a sample-sparing, robust, and reproducible manner.…”

Heterogeneity in non‐epitope loop sequence and outer membrane protein complexes alters antibody binding to the major porin protein PorB in serogroup B Neisseria meningitidis

“…The exogenous complement SBA assay was performed in 96‐well plates as previously described (Bash et al ., ) with slight modifications. Briefly, hybrid or OMP deletion strains were incubated at 37°C with shaking (65 rpm) in the presence of twofold dilutions of Z15 mAb (in 0.5% BSA in Hank's Buffered Saline Solution) and pooled human sera that had been pre‐screened for lack of endogenous killing activity.…”

Construction and protective immunogenicity of DNA vaccine pNMB0315 against Neisseria�meningitidis serogroup B

“…SBA is used to quantify the levels of antibodies that are specific for bacterial surface determinants and results in complement-mediated lysis of bacteria (25). SBA in vitro is considered as the gold standard test for the evaluation of functional anti-meningococcal antibodies and as an accepted surrogate for protection (26).…”