Abstract
Vaccines that interrupt malaria transmission are of increasing interest and a robust functional assay to measure this activity would promote their development by providing a biologically relevant means of evaluating potential vaccine candidates. Therefore, we aimed to qualify the standard membrane-feeding assay (SMFA). The assay measures the transmission-blocking activity of antibodies by feeding cultured P. falciparum gametocytes to Anopheles mosquitoes in the presence of the test antibodies and measuring subsequent mosquito infection. The International Conference on Harmonisation (ICH) Harmonised Tripartite Guideline Q2(R1) details characteristics considered in assay validation. Of these characteristics, we decided to qualify the SMFA for Precision, Linearity, Range and Specificity. The transmission-blocking 4B7 monoclonal antibody was tested over 6 feeding experiments at several concentrations to determine four suitable concentrations that were tested in triplicate in the qualification experiments (3 additional feeds) to evaluate Precision, Linearity and Range. For Specificity, 4B7 was tested in the presence of normal mouse IgG. We determined intra- and inter-assay variability of % inhibition of mean oocyst intensity at each concentration of 4B7 (lower concentrations showed higher variability). We also showed that % inhibition was dependent on 4B7 concentration and the activity is specific to 4B7. Since obtaining empirical data is time-consuming, we generated a model using data from all 9 feeds and simulated the effects of different parameters on final readouts to improve the assay procedure and analytical methods for future studies. For example, we estimated the effect of number of mosquitoes dissected on variability of % inhibition, and simulated the relationship between % inhibition in oocyst intensity and % inhibition of prevalence of infected mosquitos at different mean oocysts in the control. SMFA is one of the few biological assays used in preclinical and early clinical development of transmission-blocking vaccines, and this study strongly supports its further development and application.
Highlights
Continuous efforts to reduce malaria burden, such as application of insecticide treated nets, anti-malarial drugs and indoor insecticide spraying, have contributed to a decrease in mortality due to malaria, due to Plasmodium falciparum, from an estimated 1.8 million deaths in 2005 to 1.2 million in 2010 [1]
According to the International Conference on Harmonisation (ICH) Harmonised Tripartite Guideline Q2(R1) [8], up to seven characteristics need to be considered for assay validation depending on the type of assay: Specificity, Linearity, Range, Accuracy, Precision (Repeatability, Intermediate Precision and Reproducibility), Detection Limit, and Quantitation Limit (Table S1)
In this series of experiments, each concentration of 4B7 monoclonal antibody (mAb) was tested in a single Container of Mosquitoes (COM) in each feed
Summary
Continuous efforts to reduce malaria burden, such as application of insecticide treated nets, anti-malarial drugs and indoor insecticide spraying, have contributed to a decrease in mortality due to malaria, due to Plasmodium falciparum, from an estimated 1.8 million deaths in 2005 to 1.2 million in 2010 [1]. There is increasing interest in a transmission-blocking vaccine (TBV) which is designed to induce antibodies in human hosts against sexual stage malaria antigens or to antigens found in the mosquito vector. Several phase 1 trials have been done with TBVs, such as Plasmodium falciparum surface protein 25 (Pfs25) [3]. These existing TBV candidates are not optimal; either by inducing insufficient levels of functional antibodies in humans and/or by showing some safety concerns (the specific antigen/ adjuvant combination [not the antigen per se] was thought to cause the adverse reactions) [3]. Since an ideal TBV should induce longlasting and high levels of functional antibodies in all populations who transmit malaria, further development of effective and safe TBVs is required
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