Transmission-blocking activity is determined by transmission-reducing activity and number of control oocysts in Plasmodium falciparum standard membrane-feeding assay

Abstract

Malaria transmission-blocking vaccines (TBVs) are potentially helpful tools for malaria eradication. The standard membrane-feeding assay (SMFA) is considered one of the "gold standard" assays for TBV development. However, lack of consensus in reporting results from SMFA has made it very challenging to compare results from different studies. Two main readouts, % inhibition in mean oocyst count per mosquito (TRA) and % inhibition in prevalence of infected mosquitoes (TBA), have been used widely. In this study, we statistically modeled the oocyst data in SMFA using data from 105 independent feeding experiments including 9804 mosquitoes. The model was validated using an independent data set that included 10,790 mosquitoes from 110 feeding studies. The model delineates a relationship between TRA, the mean oocyst count in the control mosquitoes (mo-contl), and TBA. While TRA was independent from mo-contl, TBA values changed depending on mo-contl. Regardless of monoclonal or polyclonal antibodies tested, there were strong concordances between observed TBA and predicted TBA based on the model using mo-contl and observed TRA. Simulations showed that SMFA with lower true control means had increased uncertainty in TRA estimates. The strong linkage between TBA, TRA and mo-contl inspired creation of a standardized TBA, a model-based TBA standardized to a target control mean, which allows comparison across multiple feeds regardless of mo-contl. This is the first study showing that the observed TBA can be reasonably predicted by mo-contl and the TRA of the test antibody using independent experimental data. This study indicates that TRA should be used to compare results from multiple feeds with different levels of mo-contl. If a measure of TBA is desired, it is better to report standardized TBA rather than observed TBA. These recommendations support rational comparisons of results from different studies, thus benefiting future TBV development.

Keywords: Malaria; Standard membrane-feeding assay; Transmission-blocking vaccine.

Fig. 1. SMFA with fixed concentration of 4B7 mAb

Total of 104 independent feeding experiments (124 COMs) were performed with 94 μg/ml of 4B7 mAb. The results of % inhibition of mean oocyst intensity (TRA, A) and % inhibition of oocyst prevalence (TBA, B) are shown. One data point with mean oocyst in control (m_o-contl) = 0.13 and TRA = TBA =100% is not shown in the figures.

Fig. 2. Validation of the model

Independent SMFA data including 10,790 mosquitoes from 110 feeding experiments with 541 COMs were used to validate the model. The mean oocysts (x-axis in a log-scale) and prevalence (y-axis in a linear-scale) from each COM of the validation data were calculated. The data are presented with jittering and alpha blending to show overlapping points. The best-fit line (red) and the 95% prediction region (PR, blue) was estimated from the ZINB model using the model-building data (the red and blue lines are not the best-fit and the 95% PR of the validation data). The 95% PR of the model was calculated assuming 20 mosquitoes were dissected per COM (see supplement for details).

Fig. 3. Concordance between observed TBA and model-based TBA

Independent SMFA data sets (validation data) were utilized to determine whether the model could estimate TBA based on the m_o-contl and TRA of test samples. Each point represents the observed TBA (x-axis) and Model-based TBA (y-axis) with each point calibrated to the target m_t-contl that is equal to the m_o-contl of a single test sample. Data from the 4B7 mAb which targets the Pfs25 antigen (A), other mAbs targeting Pfs48/45 or Pfs230 (B) and mouse polyclonal antibodies targeting Pfs25, Pfs48/45, Pfs230, PfHAP2 or AgAPN1 (C) are shown.

Fig. 4. Number of mosquitoes to achieve the same level of error in TRA estimates

At each level of % inhibition (TRA), the number of mosquitoes that are required to achieve the same level of mean squared error in % inhibition measurement is estimated by simulation. Different colors and symbols represent different levels of TRA (0% to 95% inhibition). The reference condition is a feed where 20 mosquitoes are analyzed in each COM (20 each for control and test) and m_t-contl =10.

Fig. 5. Standardized TBA

From the 4B7 mAb data shown in Fig. 1, 17 data points were randomly selected in each specified m_o-contl range: m_o-contl were less than 6 for “low”; m_o-contl were between 6 and 30 for “med”; and m_o-contl more than 30 for “high”. Based on the observed TRA, a standardized TBA was estimated assuming m_t-contl = 2 (regardless of m_o-contl). Unstandardized TBA (A; i.e., observed TBA) and standardized TBA (B) are shown. Both individual data, the best estimate of TBA and the associated 95% confidence intervals of each group are shown. A similar figure using the full set of 4B7 mAb data, not with randomly selected data (n=17 per range in this figure), is shown in the accompanying manuscript [23] with 95% confidence intervals for individual points.