Validation of a multiplexed immunoassay for use in vaccine clinical trials and immunology studies of pre-erythrocytic malaria

Abstract Background: The primary immunological readout for clinical vaccine trials of the Plasmodium falciparum (Pf) pre-erythrocytic malaria vaccine, R21/MatrixM, is total IgG antibody specific to the central four amino acid (NANP) repeat region of the circumsporozoite protein (CSP). A multiplexed Meso Scale Discovery (MSD) assay, which includes the NANP repeat region of CSP, was developed and validated. Methods: Here we describe the validation of a multiplexed MSD assay to measure serum or plasma antibody binding to four antigens: six repeats of the NANP repeat region of P. falciparum (Pf) CSP, the C-terminus (C-term) region of CSP, full length R21 vaccine construct, and hepatitis B surface antigen (HBsAg). Initial assay optimisation, which included validation of the HBsAg international standard, was performed by MSD. Further validation performed in Oxford covered intra and inter-assay, and inter-operator variability, accuracy of QC and standard curve samples, and included bridging between the singleplex ELISA used in previous R21/MM studies, and this multiplexed assay. Results: The multiplexed MSD assay was shown to be a robust and specific assay with a broad dynamic range. We report a strong linear relationship between NANP6 IgG as measured by the singleplex ELISA and the multiplexed assay. Conclusions: This multiplexed MSD assay can be used to measure antibodies specific to the CSP NANP repeat region, CSP C-term region, full length R21 vaccine construct, and HBsAg. This assay can be used to determine humoral immune responses elicited by CSP-containing Pf malaria vaccines.