Validation of a multiplexed immunoassay for immunological analysis of pre erythrocytic malaria vaccines

Abstract

The primary immunological readout for clinical trials of R21/MatrixM™ malaria vaccine, is total IgG antibody specific to the central four amino acid NANP repeat region of the circumsporozoite protein. A multiplexed assay, which includes NANP, was developed and validated for four antigens representing components of the R21 immunogen. Initial assay optimisation included validation of the HBsAg international standard. Further validation performed in Oxford covered intra and inter-assay, and inter-operator variability, accuracy of QC and standard curve material, and included bridging to a singleplex NANP6 ELISA. The assay was shown to be robust and specific, with a broad dynamic range. We report a strong linear relationship between NANP6 IgG as measured by the singleplex ELISA and the multiplexed assay with rho values of 0.89 and 0.88 for two separate clinical trials (both p < 0.0005). This assay can be used to measure antibodies specific to the CSP NANP repeat region, CSP C-term region, full length R21 and HBsAg.

Fig. 1. Correlations between clinical trials samples run on the MSD assay in Oxford, UK and MSD, USA.

a Correlation between IgG response to full length R21 vaccine construct as measured by MSD in USA (x axis) and Oxford (y axis) using 15 samples from individuals before and following R21/MM vaccination. b Correlation between IgG response to hepatitis B surface antigen (HBsAg) as measured by MSD in USA (x-axis) and Oxford (y-axis) using 15 samples from individuals before and following R21/MM vaccination. c Correlation between IgG response to six repeats of the four amino acid sequence (NANP) as measured by MSD in USA (x-axis) and Oxford (y-axis) using 15 samples from individuals before and following R21/MM vaccination. d Correlation between IgG response to the C-terminus of CSP as measured by MSD in USA (x axis) and Oxford (y axis) using 15 samples from individuals before and following R21/MM vaccination. All figures show Spearman’s rank correlation coefficient.

Fig. 2. Concentrations for intra-run, Inter-run, and Inter-operator for each antigen included in the multiplex MSD assay. Data for 7 MSD plates used to calculate variability.

a, c, e, and g - Control (QC) samples for each of 7 plates with ± 30% thresholds for each sample. b, d, f, and h - Standards 1-7 of the standard curve for each of the 7 plates in AU/ml for full length R21 antigen, IgG in IU/mL for Hepatitis B surface antigen, IgG levels in AU/mL for NANP6 antigen, IgG levels in AU/mL for C-terminus antigen.

Fig. 3. Correlations between total IgG to NANP6 as measured by multiplexed MSD and singleplex ELISA.

a D0 (pre-vaccination samples, n = 75), D84 (1 month post primary series, n = 82) and B + 30 (1 month post booster dose given at 1 year, n = 70) for VAC076, b D84 (1 month post primary series, n = 82) and B + 30 (1-month post booster dose given at 1 year, n = 70) for VAC076. c SCRN (pre-vaccination samples, n = 23), D60 (1-month post 2 doses of R21/MM, n = 23), and D90 (1 month post primary series of 3 doses of R21/MM, n = 102) for VAC088, d D60 (1-month post 2 doses of R21/MM, n = 23), and D90 (1 month post primary series of 3 doses of R21/MM, n = 102) for VAC088. The value of 6618EU/ml is shown as a vertical line and the corresponding level measured by multiplex MSD is shown as a horizonal line for VAC076 samples only.