Evaluation of Precision of thePlasmodium knowlesiGrowth Inhibition Assay forPlasmodium vivaxDuffy-Binding Protein-based Malaria Vaccine Development

Recent data indicate increasing disease burden and importance of Plasmodium vivax ( Pv ) malaria. A robust assay will be essential for blood-stage Pv vaccine development. Results of the in vitro growth inhibition assay (GIA) with transgenic P. knowlesi ( Pk ) parasites expressing the Pv Duffy-binding protein region II ( Pv DBPII) correlate with in vivo protection in the first Pv DBPII controlled human malaria infection (CHMI) trials, making the Pk GIA an ideal selection tool once the precision of the assay is defined. To determine the precision in percentage of inhibition in GIA (%GIA) and in GIA 50 (antibody concentration that gave 50 %GIA), ten GIAs with transgenic Pk parasites were conducted evaluating four different anti- Pv DBPII human monoclonal antibodies (mAbs) at different concentrations, and three GIAs were conducted testing eighty anti- Pv DBPII human polyclonal antibodies (pAbs) at 10 mg/mL. A significant assay-to-assay variation was observed, and the analysis revealed a standard deviation (SD) of 13.1 in the mAb and 5.94 in the pAb dataset for %GIA, with a LogGIA 50 SD of 0.299 (for mAbs). Moreover, the ninety-five percent confidence interval (95%CI) for %GIA or GIA 50 in repeat assays was calculated in this investigation. These results will support the development of future blood-stage malaria vaccines, specifically second generation Pv DBPII-based formulations.