A human-serum-free medium can induce more infectious P. falciparum gametocytes than a conventional human-serum-containing medium.

Malaria remains a global health problem, and the standard membrane feeding assay (SMFA) is a key functional assay for development of new interventions to stop malaria transmission from human to mosquito. For SMFA, media with ~ 10% of human serum has been used for infectious gametocyte cultures, however, there are multiple challenges to obtain a suitable human serum. Here we show a human-serum-free culture medium (HSF), which was a mixture of two stem cell culture media and AlbuMAX, supported infectious gametocyte growth. Moreover, the HSF-induced gametocytes elicited significantly higher numbers of oocysts compared to gametocytes cultured with conventional human serum medium (Conv). While some caution is required when comparing percent transmission reducing activity data generated from HSF-SMFA and Conv-SMFA, the HSF method can facilitate the establishment of gametocyte cultures or SMFA by bypassing the need for human serum. Thus, this study will support future development of P. falciparum transmission-blocking interventions.

In Vitro Culture of Plasmodium falciparum for the Production of Mature Gametocytes for Performing Standard Membrane Feeding Assay and Infection of Anopheles spp.

Plasmodium falciparum is the parasite responsible for the disease malaria. In vitro cultivation of mature gametocytes of P. falciparum plays a central role in evaluating and developing the transmission-blocking drugs and sexual stage vaccines. These types of preventive molecules are crucial for controlling malaria in the future. Among different Plasmodium species that are involved in human malaria, only P. falciparum is cultivable. Therefore, an efficient method is required for in vitro culture of P. falciparum producing mature and infective gametocytes. This chapter describes a reliable and efficient protocol for the production of adult and infective gametocytes that is suitable for small- and large-scale culture.

Structural vaccinology of malaria transmission-blocking vaccines.

Introduction: The development of effective vaccines remains a major health priority to combat the global burden of malaria, a life-threatening disease caused by Plasmodium parasites. Transmission-blocking vaccines (TBVs) elicit antibodies that neutralize the sexual stages of the parasite in blood meals ingested by the Anopheles mosquito, interrupting parasite development in the vector host and preventing disease spread to other individuals.Areas covered: The P. falciparum gametocyte surface antigens Pfs230, Pfs48/45, and Pfs47, the parasite ookinete surface protein Pfs25, and the male gametocyte specific protein PfHAP2 are leading TBV candidates, some of which are in clinical development. The recent expansion of methodology to study monoclonal antibodies isolated directly from humans and animal models, coupled with effective measures for parasite neutralization, has provided unprecedented insight into TBV efficacy and development.Expert opinion: Available structural and functional data on antigen-monoclonal antibody (Ag-mAb) complexes, as well as epitope classification studies, have identified neutralizing epitopes that may aid vaccine development and improve protection. Here, we review the clinical prospects of TBV candidates, progress in the development of novel vaccine strategies for TBVs, and the impact of structural vaccinology in TBV design.

Strong concordance between percent inhibition in oocyst and sporozoite intensities in a Plasmodium falciparum standard membrane-feeding assay.

BACKGROUND: Effective 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. RESULTS: Plasmodium 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. CONCLUSIONS: The 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.

ELISA units, IgG subclass ratio and avidity determined functional activity of mouse anti-Pfs230 antibodies judged by a standard membrane-feeding assay with Plasmodium falciparum.

The standard membrane-feeding assay (SMFA) is a functional assay that has been used to inform the development of transmission-blocking vaccines (TBV) against Plasmodium falciparum malaria. For Pfs230, a lead target antigen for TBV development, a few studies have tested either a single anti-Pfs230 polyclonal or monoclonal antibody (one antibody per study) at serial dilutions and showed a dose-dependent response. Further, there have been reports that the SMFA activity of anti-Pfs230 polyclonal and monoclonal antibodies were enhanced in the presence of complement. However, no analysis has been performed with multiple samples, and the impact of anti-Pfs230 antibody titers, IgG subclass profile and avidity were evaluated together in relation to transmission-reducing activity (TRA) by SMFA. In this report, a total of 39 unique anti-Pfs230 IgGs from five different mouse immunization studies were assessed for their ELISA units (EU), IgG2/IgG1 ratio and avidity by ELISA, and the functionality (% transmission-reducing activity, %TRA) by SMFA. The mice were immunized with Pfs230 alone, Pfs230 conjugated to CRM197, or a mixture of unconjugated Pfs230 and CRM197 proteins using Alhydrogel or Montanide ISA720 adjuvants. In all studies, the Pfs230 antigen was from the same source. There was a significant correlation between EU and %TRA (p < 0.0001 by a Spearman rank test) for the anti-Pfs230 IgGs. Notably, multiple linear regression analyses showed that both IgG2/IgG1 ratio and avidity significantly affected %TRA (p = 0.003 to p = 0.014, depending on the models) after adjusting for EU. The results suggest that in addition to antibody titers, IgG2/IgG1 ratio and avidity should each be evaluated to predict the biological activity of anti-Pfs230 antibodies for future vaccine development.

Streamlined SMFA and mosquito dark-feeding regime significantly improve malaria transmission-blocking assay robustness and sensitivity.

BACKGROUND: The development of malaria transmission-blocking strategies including the generation of malaria refractory mosquitoes to replace the wild populations through means of gene drives hold great promise. The standard membrane feeding assay (SMFA) that involves mosquito feeding on parasitized blood through an artificial membrane system is a vital tool for evaluating the efficacy of transmission-blocking interventions. However, despite the availability of several published protocols, the SMFA remains highly variable and broadly insensitive. METHODS: The SMFA protocol was optimized through coordinated culturing of Anopheles coluzzii mosquitoes and Plasmodium falciparum parasite coupled with placing mosquitoes under a strict dark regime before, during, and after the gametocyte feed. RESULTS: A detailed description of essential steps is provided toward synchronized generation of highly fit An. coluzzii mosquitoes and P. falciparum gametocytes in preparation for an SMFA. A dark-infection regime that emulates the natural vector-parasite interaction system is described, which results in a significant increase in the infection intensity and prevalence. Using this optimal SMFA pipeline, a series of putative transmission-blocking antimicrobial peptides (AMPs) were screened, confirming that melittin and magainin can interfere with P. falciparum development in the vector. CONCLUSION: A robust SMFA protocol that enhances the evaluation of interventions targeting human malaria transmission in laboratory setting is reported. Melittin and magainin are identified as highly potent antiparasitic AMPs that can be used for the generation of refractory Anopheles gambiae mosquitoes.

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

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.