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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.

SUBMITTER: Miura K

PROVIDER: S-EPMC3590281 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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

Miura Kazutoyo K Deng Bingbing B Tullo Gregory G Diouf Ababacar A Moretz Samuel E SE Locke Emily E Morin Merribeth M Fay Michael P MP Long Carole A CA

PloS one 20130306 3

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 an ...[more]

PMID: 23483940

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Project description:BackgroundEffective malaria transmission-blocking vaccines (TBVs) can support malaria eradication programmes, and the standard membrane-feeding assay (SMFA) has been used as a "gold standard" assay for TBV development. However, in SMFA, the inhibitory activity is commonly measured at oocyst stage of parasites, while it is the sporozoites which transmit malaria from a mosquito to a human. A handful of studies have shown that there is a positive correlation between oocyst and sporozoite intensities. However, no study has been completed to compare inhibition levels in oocyst and sporozoite intensities in the presence of transmission-blocking (TB) antibodies.ResultsPlasmodium falciparum NF54 gametocytes were fed to Anopheles stephensi mosquitoes with or without anti-Pfs25 or anti-Pfs48/45 TB antibodies in 15 independent assays. For each group, a portion of the mosquitoes was dissected for oocyst counts (day 8 after feed), and a portion of the remaining mosquitoes was dissected for sporozoite counts (day 16). This study covered a large range of oocyst and sporozoite intensities: 0.2 to 80.5 on average for oocysts, and 141 to 77,417 for sporozoites. The sporozoite data were well explained by a zero-inflated negative binomial model, regardless of the presence or absence of TB antibodies. Inhibition levels in both oocyst and sporozoite intensities were determined within the same groups in 9 independent assays. When the level of inhibition in sporozoite number (expressed as Log Mean Ratio, LMR; average number in a control group was divided by the one in a test group, then took a log of the ratio) was plotted against LMR in oocyst number, the best-fit slope of a linear regression was not different from 1 (the best estimate, 1.08; 95% confidence interval, 0.87 to 1.29). Furthermore, a Bland-Altman analysis showed a strong agreement between inhibitions in oocysts and in sporozoites.ConclusionsThe results indicate that percent inhibition in oocyst intensity of a test sample can be directly converted to % inhibition in sporozoite intensity in P. falciparum SMFA. Therefore, if sporozoite intensity determines transmission rate from mosquitoes to humans, the percent inhibition in oocyst intensity measured by SMFA can be used to estimate the TBV efficacy.

Project description:BackgroundMosquito feeding assays using venous blood are commonly used for evaluating the transmission potential of malaria infected individuals. To improve the accuracy of these assays, care must be taken to prevent premature activation or inactivation of gametocytes before they are fed to mosquitoes. This can be challenging in the field where infected individuals and insectary facilities are sometimes very far apart. In this study, a simple, reliable, field applicable method is presented for storage and transport of gametocyte infected blood using a thermos flask.MethodsThe optimal storage conditions for maintaining the transmissibility of gametocytes were determined initially using cultured Plasmodium falciparum gametocytes in standard membrane feeding assays (SMFAs). The impact of both the internal thermos water temperature (35.5 to 37.8 °C), and the external environmental temperature (room temperature to 42 °C) during long-term (4 h) storage, and the impact of short-term (15 min) temperature changes (room temp to 40 °C) during membrane feeding assays was assessed. The optimal conditions were then evaluated in direct membrane feeding assays (DMFAs) in Burkina Faso and The Gambia where blood from naturally-infected gametocyte carriers was offered to mosquitoes immediately and after storage in thermos flasks.ResultsUsing cultured gametocytes in SMFAs it was determined that an internal thermos water temperature of 35.5 °C and storage of the thermos flask between RT (~ 21.3 °C) and 32 °C was optimal for maintaining transmissibility of gametocytes for 4 h. Short-term storage of the gametocyte infected blood for 15 min at temperatures up to 40 °C (range: RT, 30 °C, 38 °C and 40 °C) did not negatively affect gametocyte infectivity. Using samples from natural gametocyte carriers (47 from Burkina Faso and 16 from The Gambia), the prevalence of infected mosquitoes and the intensity of oocyst infection was maintained when gametocyte infected blood was stored in a thermos flask in water at 35.5 °C for up to 4 h.ConclusionsThis study determines the optimal long-term (4 h) storage temperature for gametocyte infected blood and the external environment temperature range within which gametocyte infectivity is unaffected. This will improve the accuracy, reproducibility, and utility of DMFAs in the field, and permit reliable comparative assessments of malaria transmission epidemiology in different settings.

Dataset Information

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

Publications

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

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