Qualification of standard membrane-feeding assay with Plasmodium falciparum malaria and potential improvements for future assays

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.

Figure 1. Dose dependent % inhibition in mean oocyst intensity by 4B7 mAb.

Various concentrations of 4B7 mAbs (ranging from 1 to 375 µg/ml) were tested over 6 independent feeding experiments (Feed 1–6). Different symbols represent data from different feeding experiments.

Figure 2. Intra- and inter-feed variability in PIm of 4B7 mAb.

Four concentrations (1, 6, 23 and 94 µg/ml) of 4B7 mAb were tested in triplicate in each feed, and three independent feeds were performed (Feed 7, 8 and 9). Since there were 3 COM of negative control and 3 COM of 4B7 mAb at each concentration, 9 different numbers of PIm were calculated (individual dots) at each concentration in each feed. Bar represents the mean of the 9 calculated data.

Figure 3. Relationship between 4B7 concentration and PIm.

Various concentrations of 4B7 mAb were tested in the qualification experiments (Feed 7–9). For these data the first COM negative control is matched with the first COM of the 4B7 mAb at each concentration, the second with the second, etc. The square root of 4B7 concentration is shown on the x-axis, and the ratio of mean oocyst (mean of oocysts in control divided by mean of oocysts in test) is plotted on a log scale (shown on left side of y-axis, the associated PIm value is shown on the right side of the y-axis). Points with the same symbol use the same control, and points with the same color are from the same feed. Dotted line represents the best-fit line.

Figure 4. Effect of normal mouse IgG on 4B7 mAb.

Two concentrations (23 and 94 µg/ml) of 4B7 mAb were tested with or without 0.75 mg/ml of normal mouse IgG (NMAb). PIm without NMAb were calculated 3 times for each feed (using 3 test COM and 3 separate control COM, 20 mosquitoes in each COM) and 1 time (1 test COM and 1 control COM, 20 mosquitoes each) for PIm with NMAb.

Figure 5. Relationship between mean number of oocysts and the standard deviation.

For each COM, mean number of oocysts and standard deviation were calculated. Data from all COM tested in 9 independent feeding experiments are shown. Different symbols represent data from different feeding experiments and the line represents the best-fit curve from the zero-inflated negative binomial model. The R² value for the fit is 0.94. Gray lines represent 95% confidence intervals calculated using the t-distribution (for the means) or chi square distribution (for the standard deviations).

Figure 6. Effect of modifications of assay on the sensitivity of SMFA.

In this simulation, we assumed there were two test samples (T₁ and T₂), and true PIm of T₁ (50 or 70% inhibition compared to control) was higher than the true PIm of T₂ (0, 10, 20, 30, 40 or 50%). Three different dissection conditions were simulated; 1) total of 20 mosquitoes were dissected from a single COM (m = 20), 2) total of 60 from a single COM (m = 60), and 3) total of 60, but from three COM (m = 20×3). In addition, we stimulated either: 1) T₁ and T₂ were tested in the same feeding experiment (SF), or 2) tested in different feeding experiments (DF). We assumed the mean number of oocysts in the control was 20. For each test condition, 10,000 data were generated to calculate the probability of feeds in which T₁ showed higher PIm (i.e., lower mean oocyst number) than that T₂.

Figure 7. Sample mean of oocyst counts by proportion of mosquitoes with any infection.

Each point represents one COM. Black line is the fit from the zero-inflated negative binomial model. The blue dotted line is a nonparametric moving window average (specifically, a kernel smoother with a normal kernel with bandwidth 0.5 log₁₀ chosen to be slightly overfit).

Figure 8. Effect of mean number of oocysts in the control on the two % inhibitions.

The % inhibition of prevalence (PIp) is plotted against % inhibition in mean oocyst intensity (PIm) at different mean number of oocysts in the control.