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An inter-laboratory comparison of standard membrane-feeding assays for evaluation of malaria transmission-blocking vaccines

View Article: PubMed Central - PubMed

ABSTRACT

Background: An effective malaria transmission-blocking vaccine may play an important role in malaria elimination efforts, and a robust biological assay is essential for its development. The standard membrane-feeding assay (SMFA) for Plasmodium falciparum infection of mosquitoes is considered a “gold standard” assay to measure transmission-blocking activity of test antibodies, and has been utilized widely in both non-clinical and clinical studies. While several studies have discussed the inherent variability of SMFA within a study group, there has been no assessment of inter-laboratory variation. Therefore, there is currently no assurance that SMFA results are comparable between different studies.

Methods: Mouse anti-Pfs25 monoclonal antibody (mAb, 4B7 mAb), rat anti-Pfs48/45 mAb (85RF45.1 mAb) and a human polyclonal antibody (pAb) collected from a malaria-exposed adult were tested at the same concentrations (6–94 μg/mL for 4B7, 1.2–31.3 μg/mL for 85RF45.1 and 23–630 μg/mL for human pAb) in two laboratories following their own standardized SMFA protocols. The mAbs and pAb, previously shown to have strong inhibition activities in the SMFA, were tested at three or four concentrations in two or three independent assays in each laboratory, and percent inhibition in mean oocyst intensity relative to a control in the same feed was determined in each feeding experiment.

Results: Both monoclonal and polyclonal antibodies dose-dependently reduced oocyst intensity in all experiments performed at the two test sites. In both laboratories, the inter-assay variability in percent inhibition in oocyst intensity decreased at higher levels of inhibition, regardless of which antibody was tested. At antibody concentrations that led to a >80 % reduction in oocyst numbers, the inter-laboratory variations were in the same range compared with the inter-assay variation observed within a single laboratory, and the differences in best estimates from multiple feeds between the two laboratories were <5 percentage points.

Conclusions: This study confirms previous reports that the precision of the SMFA increases with increasing percent inhibition. Moreover, the variation between the two laboratories is not greater than the variation observed within a laboratory. The findings of this study provide guidance for comparison of SMFA data from different laboratories.

Electronic supplementary material: The online version of this article (doi:10.1186/s12936-016-1515-z) contains supplementary material, which is available to authorized users.

No MeSH data available.


SMFA comparison with 85RF45.1 mAb. In both laboratories, rat 85RF45.1 mAb was tested at 1.2, 3.5, 10.4 and 31.3 μg/mL in two feeding experiments (F1 and F2). The individual data and the best estimate with 95 % CIs from the two feeds (LMVR-Comb or TropIQ-Comb) at each concentration of 85RF45.1 mAb are shown. Average oocysts in a negative control antibody (normal mouse IgG) of LMVR-F1, -F2, TropIQ-F1, and -F2 experiments were 7.8, 61.6, 6.5 and 4.2, respectively
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Fig3: SMFA comparison with 85RF45.1 mAb. In both laboratories, rat 85RF45.1 mAb was tested at 1.2, 3.5, 10.4 and 31.3 μg/mL in two feeding experiments (F1 and F2). The individual data and the best estimate with 95 % CIs from the two feeds (LMVR-Comb or TropIQ-Comb) at each concentration of 85RF45.1 mAb are shown. Average oocysts in a negative control antibody (normal mouse IgG) of LMVR-F1, -F2, TropIQ-F1, and -F2 experiments were 7.8, 61.6, 6.5 and 4.2, respectively

Mentions: Rat 85RF45.1 (anti-Pfs48/45) mAb was next tested at four different concentrations in two independent assays in both laboratories. The mAb dose-dependently inhibited oocyst formation. At higher concentrations of the mAb (i.e. 10.4 and 31.3 μg/mL), the %TRA observed for replicate experiments within labs and between labs were very comparable (Fig. 3). At lower concentrations, the observed %TRA diverged more between replicate experiments, both within and between laboratories (i.e. concentrations of 1.2 and 3.5 μg/mL). Since four different doses of mAb were tested in each feed, and the same number of feeds were conducted in each laboratory, further analysis was performed to determine the effects of dose and laboratory on inhibition. In a multiple linear regression analysis, dose of 85RF45.1 mAb, laboratory, and the interaction of the two were used as explanatory variables. The overall fit to the linear regression model was R2 = 0.73. While there was a significant effect of dose (p = 0.032), the laboratory (p = 0.427) and interaction term (dose and laboratory, p = 0.659) had no significant impact. The effect of dose on oocyst intensity was further investigated by fitting the data to a Hill equation (see Additional file 1). Resulting IC50 estimates (the concentration at which the mAb shows 50 % inhibition) were 1.2 and 8.8 µg/mL [feed 1 (F1) and feed 2 (F2) experiments] for data generated at LMVR and 3.4 and 2.5 µg/mL (F1 and F2) for data generated at TropIQ.Fig. 3


An inter-laboratory comparison of standard membrane-feeding assays for evaluation of malaria transmission-blocking vaccines
SMFA comparison with 85RF45.1 mAb. In both laboratories, rat 85RF45.1 mAb was tested at 1.2, 3.5, 10.4 and 31.3 μg/mL in two feeding experiments (F1 and F2). The individual data and the best estimate with 95 % CIs from the two feeds (LMVR-Comb or TropIQ-Comb) at each concentration of 85RF45.1 mAb are shown. Average oocysts in a negative control antibody (normal mouse IgG) of LMVR-F1, -F2, TropIQ-F1, and -F2 experiments were 7.8, 61.6, 6.5 and 4.2, respectively
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC5016893&req=5

Fig3: SMFA comparison with 85RF45.1 mAb. In both laboratories, rat 85RF45.1 mAb was tested at 1.2, 3.5, 10.4 and 31.3 μg/mL in two feeding experiments (F1 and F2). The individual data and the best estimate with 95 % CIs from the two feeds (LMVR-Comb or TropIQ-Comb) at each concentration of 85RF45.1 mAb are shown. Average oocysts in a negative control antibody (normal mouse IgG) of LMVR-F1, -F2, TropIQ-F1, and -F2 experiments were 7.8, 61.6, 6.5 and 4.2, respectively
Mentions: Rat 85RF45.1 (anti-Pfs48/45) mAb was next tested at four different concentrations in two independent assays in both laboratories. The mAb dose-dependently inhibited oocyst formation. At higher concentrations of the mAb (i.e. 10.4 and 31.3 μg/mL), the %TRA observed for replicate experiments within labs and between labs were very comparable (Fig. 3). At lower concentrations, the observed %TRA diverged more between replicate experiments, both within and between laboratories (i.e. concentrations of 1.2 and 3.5 μg/mL). Since four different doses of mAb were tested in each feed, and the same number of feeds were conducted in each laboratory, further analysis was performed to determine the effects of dose and laboratory on inhibition. In a multiple linear regression analysis, dose of 85RF45.1 mAb, laboratory, and the interaction of the two were used as explanatory variables. The overall fit to the linear regression model was R2 = 0.73. While there was a significant effect of dose (p = 0.032), the laboratory (p = 0.427) and interaction term (dose and laboratory, p = 0.659) had no significant impact. The effect of dose on oocyst intensity was further investigated by fitting the data to a Hill equation (see Additional file 1). Resulting IC50 estimates (the concentration at which the mAb shows 50 % inhibition) were 1.2 and 8.8 µg/mL [feed 1 (F1) and feed 2 (F2) experiments] for data generated at LMVR and 3.4 and 2.5 µg/mL (F1 and F2) for data generated at TropIQ.Fig. 3

View Article: PubMed Central - PubMed

ABSTRACT

Background: An effective malaria transmission-blocking vaccine may play an important role in malaria elimination efforts, and a robust biological assay is essential for its development. The standard membrane-feeding assay (SMFA) for Plasmodium falciparum infection of mosquitoes is considered a “gold standard” assay to measure transmission-blocking activity of test antibodies, and has been utilized widely in both non-clinical and clinical studies. While several studies have discussed the inherent variability of SMFA within a study group, there has been no assessment of inter-laboratory variation. Therefore, there is currently no assurance that SMFA results are comparable between different studies.

Methods: Mouse anti-Pfs25 monoclonal antibody (mAb, 4B7 mAb), rat anti-Pfs48/45 mAb (85RF45.1 mAb) and a human polyclonal antibody (pAb) collected from a malaria-exposed adult were tested at the same concentrations (6–94 μg/mL for 4B7, 1.2–31.3 μg/mL for 85RF45.1 and 23–630 μg/mL for human pAb) in two laboratories following their own standardized SMFA protocols. The mAbs and pAb, previously shown to have strong inhibition activities in the SMFA, were tested at three or four concentrations in two or three independent assays in each laboratory, and percent inhibition in mean oocyst intensity relative to a control in the same feed was determined in each feeding experiment.

Results: Both monoclonal and polyclonal antibodies dose-dependently reduced oocyst intensity in all experiments performed at the two test sites. In both laboratories, the inter-assay variability in percent inhibition in oocyst intensity decreased at higher levels of inhibition, regardless of which antibody was tested. At antibody concentrations that led to a >80 % reduction in oocyst numbers, the inter-laboratory variations were in the same range compared with the inter-assay variation observed within a single laboratory, and the differences in best estimates from multiple feeds between the two laboratories were <5 percentage points.

Conclusions: This study confirms previous reports that the precision of the SMFA increases with increasing percent inhibition. Moreover, the variation between the two laboratories is not greater than the variation observed within a laboratory. The findings of this study provide guidance for comparison of SMFA data from different laboratories.

Electronic supplementary material: The online version of this article (doi:10.1186/s12936-016-1515-z) contains supplementary material, which is available to authorized users.

No MeSH data available.