Murine Model for Assessment of Plasmodium falciparum Transmission-Blocking Vaccine Using Transgenic Plasmodium berghei Parasites Expressing the Target Antigen Pfs25

Abstract

Currently, there is no animal model for Plasmodium falciparum challenge to evaluate malaria transmissionblocking vaccines based on the well-established Pfs25 target antigen. The biological activity of transmissionblocking antibodies is typically assessed using an assay known as the membrane feeding assay (MFA). It is an in vitro method that involves mixing antibodies with cultured P. falciparum gametocytes and feeding them to mosquitoes through an artificial membrane followed by assessment of infection in the mosquitoes. We genetically modified Plasmodium berghei to express Pfs25 and demonstrated that the transgenic parasites (TrPfs25Pb) are susceptible to anti-Pfs25 antibodies during mosquito-stage development. The asexual growth kinetics and mosquito infectivity of TrPfs25Pb were comparable to those of wild-type parasites, and TrPfs25Pb displayed Pfs25 on the surface of ookinetes. Immune sera from nonhuman primates immunized with a Pfs25-based vaccine when passively transferred to mice blocked transmission of TrPfs25Pb to Anopheles stephensi. Furthermore, mice immunized with Pfs25 DNA vaccine and challenged with TrPfs25Pb displayed reduced malaria transmission compared to mice immunized with wild-type plasmid. These studies describe development of an animal malaria model alternative to the in vitro MFA and show that the model can facilitate P. falciparum transmission-blocking vaccine evaluation based on the target antigen Pfs25. We believe that an animal model to test transmission-blocking vaccines would be superior to the MFA, since there may be additional immune factors that synergize the transmission-blocking activity of antibodies in vivo.Annually, 300 to 500 million people worldwide suffer from clinical malaria, and approximately 1 million people, mainly children under 5 years old, die as a result of the disease (8, 18). Successful transmission of Plasmodium parasites relies on the uptake of sexual stages (gametocytes) of the pathogen by mosquitoes during a blood meal. Subsequent gametogenesis and fertilization of female and male gametes establishes a sexual reproduction phase in the mosquito midgut. Resulting zygotes transform into motile ookinetes, which traverse the peritrophic matrix and midgut epithelium, lodge on the basal lamina of the midgut, and develop into oocysts. Sporozoites produced in the oocysts are released into the hemocoel, then invade salivary glands, and are subsequently introduced to new hosts when the mosquito takes another blood meal. A number of stage-specific proteins have been shown to play important roles during fertilization and ookinete formation, and antibodies recognizing these antigens are potent blockers of parasite development in mosquitoes (12). Among the proteins that are expressed on the surface of zygotes and ookinetes, P25 and P28 have been shown to be crucial for successful transmission of Plasmodium parasites (7, 21). Both P25 and P28 are strong candidates for transmission-blocking vaccines (TBV), and phase I clinical trials for Pfs25 (Plasmodium falciparum) and P...

Transgenic Rodent Plasmodium berghei Parasites as Tools for Assessment of Functional Immunogenicity and Optimization of Human Malaria Vaccines

“…When passively transferred into mice, immune sera from nonhuman primates (rhesus monkeys) immunized with a Pfs25-based vaccine blocked the transmission of TrPfs25Pb to Anopheles stephensi by a magnitude of at least 90%. Moreover, mice immunized with a Pfs25 DNA vaccine and challenged with TrPfs25Pb displayed reduced malaria transmission compared to mice immunized with a control wild-type plasmid (23). The results of these studies have established that murine malaria models can serve as an alternative to in vitro MFA for the evaluation of P. falciparum or P. vivax TBV based on the target antigen P25.…”

“…Subsequently, Pv25DR and Pv25DR3 were employed to test the transmission-blocking potential of human sera from a clinical trial based on Pvs25 (29). In parallel, transgenic P. berghei parasites expressing P. falciparum P25 (Pfs25) have also been developed, and these transgenic parasites, designated TrPfs25Pb, were found to be susceptible to anti-Pfs25 antibodies during mosquito-stage development (23). TrPfs25Pb parasites displayed Pfs25 on the surfaces of zygotes and ookinetes.…”

“…The results of these studies have established that murine malaria models can serve as an alternative to in vitro MFA for the evaluation of P. falciparum or P. vivax TBV based on the target antigen P25. Further studies to define the stage specificities of transmissionblocking antibodies were also attempted using an in vitro ookinete assay with Pv25DR, Pv25DR3, and TrPfs25Pb parasites (23,29). In this assay, blood-stage parasites are cultured to produce ookinetes (a motile stage of the parasite formed after zygote formation) in the presence of antibodies to assess transmission-blocking activity, and the magnitude of blocking varied from 50 to about 70% (23,29).…”

Development of a Chimeric Plasmodium berghei Strain Expressing the Repeat Region of the P. vivax Circumsporozoite Protein forIn VivoEvaluation of Vaccine Efficacy

“…Human studies are expensive, and therefore, this is not a practical first step to evaluate vaccine candidates. Over the last decade or so, transfection of Plasmodium has revolutionized the ways in which protective immune responses can be evaluated using transgenic rodent parasites that have been transfected with the gene encoding a specific human malaria antigen (14,(26)(27)(28)(29). Active-and passive-immunization studies directed toward these antigens are more indicative of a biologically relevant immune response than the evaluation of immunogenicity alone.…”

“…Active-and passive-immunization studies directed toward these antigens are more indicative of a biologically relevant immune response than the evaluation of immunogenicity alone. Transgenic parasites carrying the CSP, MSP, and P25 genes of P. falciparum have been developed and are being used to evaluate vaccine candidates and/or platforms (14,(26)(27)(28)(29). To date, only one transgenic P. berghei sporozoite expressing P25 from P. vivax has been developed as a tool for evaluating the efficacy of vaccine candidates (29).…”