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-TT-primed 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.

FIG 1

hSBA titers of serum samples determined using independent pooled human complement (hC′) lots. Two-fold serum dilutions from 1:4 to 1:1,024 were tested. Titers of <4 are shown as values of 2. (A) Comparison of hSBA titers for 15 samples using hC′ lots C12, C13, and C14 analyzed in the CBER laboratory; data for 3 samples tested with lot C11 are included. Indeterminate titers (n = 2) and titers of ≥64 with insufficient sample to test at higher dilutions (n = 2) are not shown. (B) Comparison of reported titers for 18 samples tested using hC′ lots C11, C12, and C13 in the PHE laboratory. Two samples with data available for only one hC′ lot are not shown. (C) Comparison of the median values of 14 samples between the CBER and PHE laboratories. Sample 33005 is not shown, as a valid median titer (panel B) was not determined.

FIG 2

The hSBA geometric mean titers (GMTs) are shown by vaccination group at 28 days and 10 months after primary immunization (H, Hib; M, PsACWY; P, PsA-TT). For groups that received a second meningococcal immunization, GMTs are shown at 28 days after the second immunization and at 2 years postenrollment. The first letter designates the first immunization, and the second letter designates the second immunization. Error bars show ±1 standard deviation.

FIG 3

Reverse cumulative distribution curves for hSBA responses to PsA-TT vaccination. Shown is the proportion of subjects with hSBA titers at or above the x axis value for the PsA-TT primary immunization group at 28 days (P 1 mo) and 10 months (P 10 mo) after primary immunization, PsA-TT-primed subjects who received a second PsA-TT dose (PP) or a one-fifth dose of PsACWY challenge (PM) at 28 days (PP 11 mo or PM 11 mo, respectively) and 14 months (PP 24 mo or PM 24 mo, respectively) after the second immunization. The distribution of hSBA titers among the reference PsACWY vaccine group is shown at 28 days after primary immunization (M 1 mo).