C-type lectin 4 regulates broad-spectrum melanization-based refractoriness to malaria parasites

Anopheles gambiae melanization-based refractoriness to the human malaria parasite Plasmodium falciparum has rarely been observed in either laboratory or natural conditions, in contrast to the rodent model malaria parasite Plasmodium berghei that can become completely melanized by a TEP1 complement-like system-dependent mechanism. Multiple studies have shown that the rodent parasite evades this defense by recruiting the C-type lectins CTL4 and CTLMA2, while permissiveness to the human malaria parasite was not affected by partial depletion of these factors by RNAi silencing. Using CRISPR/Cas9-based CTL4 knockout, we show that A. gambiae can mount melanization-based refractoriness to the human malaria parasite, which is independent of the TEP1 complement-like system and the major anti-Plasmodium immune pathway Imd. Our study indicates a hierarchical specificity in the control of Plasmodium melanization and proves CTL4 as an essential host factor for P. falciparum transmission and one of the most potent mosquito-encoded malaria transmission-blocking targets

Clinically relevant atovaquone-resistant human malaria parasites fail to transmit by mosquito.

Long-acting injectable medications, such as atovaquone, offer the prospect of a "chemical vaccine" for malaria, combining drug efficacy with vaccine durability. However, selection and transmission of drug-resistant parasites is of concern. Laboratory studies have indicated that atovaquone resistance disadvantages parasites in mosquitoes, but lack of data on clinically relevant Plasmodium falciparum has hampered integration of these variable findings into drug development decisions. Here we generate atovaquone-resistant parasites that differ from wild type parent by only a Y268S mutation in cytochrome b, a modification associated with atovaquone treatment failure in humans. Relative to wild type, Y268S parasites evidence multiple defects, most marked in their development in mosquitoes, whether from Southeast Asia (Anopheles stephensi) or Africa (An. gambiae). Growth of asexual Y268S P. falciparum in human red cells is impaired, but parasite loss in the mosquito is progressive, from reduced gametocyte exflagellation, to smaller number and size of oocysts, and finally to absence of sporozoites. The Y268S mutant fails to transmit from mosquitoes to mice engrafted with human liver cells and erythrocytes. The severe-to-lethal fitness cost of clinically relevant atovaquone resistance to P. falciparum in the mosquito substantially lessens the likelihood of its transmission in the field

A modified Plasmodium falciparum growth inhibition assay (GIA) to assess activity of plasma from malaria endemic areas

Plasma samples from patients undergoing treatment in malaria endemic countries often contain anti-malaria drugs, that may overstate effects of specific antibodies in growth inhibition assays (GIA). We describe a modified assay that uses drug resistant P. falciparum parasites (W2) that circumvents the requirement for dialyzing samples that may likely contain drugs such as chloroquine and sulfadoxine/pyrimethamine (SP). © 2006 Elsevier Inc. All rights reserved

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

Currently, there is no animal model for Plasmodium falciparum challenge to evaluate malaria transmission-blocking vaccines based on the well-established Pfs25 target antigen. The biological activity of transmission-blocking 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

Aberrant Sporogonic Development of Dmc1 (a Meiotic Recombinase) Deficient Plasmodium berghei Parasites

Background: In Plasmodium, meiosis occurs in diploid zygotes as they develop into haploid motile ookinetes inside the mosquito. Further sporogonic development involves transformation of ookinetes into oocysts and formation of infective sporozoites. Methodology/Principal Findings: Reverse genetics was employed to examine the role of the meiotic specific recombinase Dmc1, a bacterial RecA homolog during sporogony in Plasmodium berghei. PbDmc1 knockout (KO) parasites showed normal asexual growth kinetics compared to WT parasites; however oocyst formation in mosquitoes was reduced by 50 to 80%. Moreover, the majority of oocysts were retarded in their growth and were smaller in size compared to WT parasites. Only a few Dmc1 KO parasites completed maturation resulting in formation of fewer sporozoites which were incapable of infecting naive mice or hepatocytes in vitro. PbDmc1 KO parasites were shown to be approximately 18 times more sensitive to Bizelesin, a DNA alkylating drug compared to WT parasites as reflected by impairment of oocyst formation and sporogonic development in the mosquito vector. Conclusions/Significance: Our findings suggest that PbDmc1 plays a critical role in malaria transmission biology. © 2012 Mlambo et al

Functional immunogenicity of baculovirus expressing Pfs25, a human malaria transmission-blocking vaccine candidate antigen

We have focused on development of a novel vaccine vector based on Baculophage , a baculovirus display system for expression of proteins on the surface of the viral envelope, as a non-pathogenic and non-vertebrate insect virus. In the present study, recombinant baculovirus (AcNPV-Pfs25surf) were generated, which displayed Pfs25, a potent Plasmodium falciparum transmission-blocking vaccine candidate. Both intranasal and intramuscular immunizations of mice with AcNPV-Pfs25surf induced high levels of Pfs25-specific antibodies, which strongly reacted with ookinetes of transgenic Plasmodium berghei expressing Pfs25 (TrPfs25Pb). Importantly, sera obtained from immunized rabbits exhibited a significant transmission-blocking effect (\u3e90% reduction in infection intensity) in standard membrane feeding assay using P. falciparum gametocytes. Additionally, active immunization (both intranasal and intramuscular routes) of mice followed by challenge using TrPfs25Pb demonstrated an effective transmission-blocking response, with an 83% (intranasal) and ∼95% (intramuscular) reduction in oocyst intensity, respectively. Thus, the baculovirus-based vaccines offer a promising new alternative to current human vaccine delivery platforms for the development of malaria multi-stage vaccines. © 2010 Elsevier Ltd