Quantification of female and male Plasmodium falciparum gametocytes by reverse transcriptase quantitative PCR

The transmission of malaria parasites depends on the presence of sexual stages (gametocytes) in the blood, making the ratio and densities of female and male gametocytes important determinants of parasite fitness. This manuscript describes the development of reverse transcriptase quantitative PCR (RT-qPCR) assays to separately quantify mature female and male gametocytes of the human malaria parasite Plasmodium falciparum, and reveals that Pfs25 mRNA is expressed only in female gametocytes. The female (Pfs25) and male (Pfs230p) gametocyte specific RT-qPCR assays have lower detection limits of 0.3 female and 1.8 male gametocytes per microlitre of blood, respectively, making them more sensitive than microscopy. Accurate quantification of the ratio and densities of female and male gametocytes will increase understanding of P. falciparum transmission and improve the evaluation of transmission blocking interventions

Removal of Heterologous Sequences from Plasmodium falciparum Mutants Using FLPe-Recombinase

Genetically-modified mutants are now indispensable Plasmodium gene-function reagents, which are also being pursued as genetically attenuated parasite vaccines. Currently, the generation of transgenic malaria-parasites requires the use of drug-resistance markers. Here we present the development of an FRT/FLP-recombinase system that enables the generation of transgenic parasites free of resistance genes. We demonstrate in the human malaria parasite, P. falciparum, the complete and efficient removal of the introduced resistance gene. We targeted two neighbouring genes, p52 and p36, using a construct that has a selectable marker cassette flanked by FRT-sequences. This permitted the subsequent removal of the selectable marker cassette by transient transfection of a plasmid that expressed a 37°C thermostable and enhanced FLP-recombinase. This method of removing heterologous DNA sequences from the genome opens up new possibilities in Plasmodium research to sequentially target multiple genes and for using genetically-modified parasites as live, attenuated malaria vaccines

Gene Disruption of Plasmodium falciparum p52 Results in Attenuation of Malaria Liver Stage Development in Cultured Primary Human Hepatocytes

Difficulties with inducing sterile and long lasting protective immunity against malaria with subunit vaccines has renewed interest in vaccinations with attenuated Plasmodium parasites. Immunizations with sporozoites that are attenuated by radiation (RAS) can induce strong protective immunity both in humans and rodent models of malaria. Recently, in rodent parasites it has been shown that through the deletion of a single gene, sporozoites can also become attenuated in liver stage development and, importantly, immunization with these sporozoites results in immune responses identical to RAS. The promise of vaccination using these genetically attenuated sporozoites (GAS) depends on translating the results in rodent malaria models to human malaria. In this study, we perform the first essential step in this transition by disrupting, p52, in P. falciparum an ortholog of the rodent parasite gene, p36p, which we had previously shown can confer long lasting protective immunity in mice. These P. falciparum P52 deficient sporozoites demonstrate gliding motility, cell traversal and an invasion rate into primary human hepatocytes in vitro that is comparable to wild type sporozoites. However, inside the host hepatocyte development is arrested very soon after invasion. This study reveals, for the first time, that disrupting the equivalent gene in both P. falciparum and rodent malaria Plasmodium species generates parasites that become similarly arrested during liver stage development and these results pave the way for further development of GAS for human use

Three Members of the 6-cys Protein Family of Plasmodium Play a Role in Gamete Fertility

The process of fertilization is critically dependent on the mutual recognition of gametes and in Plasmodium, the male gamete surface protein P48/45 is vital to this process. This protein belongs to a family of 10 structurally related proteins, the so called 6-cys family. To identify the role of additional members of this family in Plasmodium fertilisation, we performed genetic and functional analysis on the five members of the 6-cys family that are transcribed during the gametocyte stage of P. berghei. This analysis revealed that in addition to P48/45, two members (P230 and P47) also play an essential role in the process of parasite fertilization. Mating studies between parasites lacking P230, P48/45 or P47 demonstrate that P230, like P48/45, is a male fertility factor, consistent with the previous demonstration of a protein complex containing both P48/45 and P230. In contrast, disruption of P47 results in a strong reduction of female fertility, while males remain unaffected. Further analysis revealed that gametes of mutants lacking expression of p48/45 or p230 or p47 are unable to either recognise or attach to each other. Disruption of the paralog of p230, p230p, also specifically expressed in gametocytes, had no observable effect on fertilization. These results indicate that the P. berghei 6-cys family contains a number of proteins that are either male or female specific ligands that play an important role in gamete recognition and/or attachment. The implications of low levels of fertilisation that exist even in the absence of these proteins, indicating alternative pathways of fertilisation, as well as positive selection acting on these proteins, are discussed in the context of targeting these proteins as transmission blocking vaccine candidates

Protection against a malaria challenge by sporozoite inoculation.

Contains fulltext : 81826.pdf (publisher's version ) (Open Access)BACKGROUND: An effective vaccine for malaria is urgently needed. Naturally acquired immunity to malaria develops slowly, and induction of protection in humans can be achieved artificially by the inoculation of radiation-attenuated sporozoites by means of more than 1000 infective mosquito bites. METHODS: We exposed 15 healthy volunteers--with 10 assigned to a vaccine group and 5 assigned to a control group--to bites of mosquitoes once a month for 3 months while they were receiving a prophylactic regimen of chloroquine. The vaccine group was exposed to mosquitoes that were infected with Plasmodium falciparum, and the control group was exposed to mosquitoes that were not infected with the malaria parasite. One month after the discontinuation of chloroquine, protection was assessed by homologous challenge with five mosquitoes infected with P. falciparum. We assessed humoral and cellular responses before vaccination and before the challenge to investigate correlates of protection. RESULTS: All 10 subjects in the vaccine group were protected against a malaria challenge with the infected mosquitoes. In contrast, patent parasitemia (i.e., parasites found in the blood on microscopical examination) developed in all five control subjects. Adverse events were mainly reported by vaccinees after the first immunization and by control subjects after the challenge; no serious adverse events occurred. In this model, we identified the induction of parasite-specific pluripotent effector memory T cells producing interferon-gamma, tumor necrosis factor alpha, and interleukin-2 as a promising immunologic marker of protection. CONCLUSIONS: Protection against a homologous malaria challenge can be induced by the inoculation of intact sporozoites. (ClinicalTrials.gov number, NCT00442377.

Generation of <i>P. falciparum</i> parasites lacking expression of P52.

<p>(A) Illustration of the DNA construct (m144) used for the targeted gene disruption of <i>p52</i> and the <i>p52</i>-genomic locus before and after integration. Shown are the p52 gene and target sequence (amplified using 1624 & 1625), the paralog of p52, p36, and the <i>T. gondii dhfr/ts</i> selection cassette. In addition, primer pairs and restriction sites for diagnostic PCR and Southern analysis are shown (see B and C). hrp – histidine rich protein. (B) Southern analysis of <i>BstN</i>I/<i>SnaB</i>I digested genomic DNA of Wt and <i>Δp52</i> demonstrates correct disruption of <i>p52</i>. DNA was hybridized with a <i>p52</i> specific probe detecting a 3.3 kb fragment in Wt, a 2.2 kb fragment for intact plasmid and the expected fragments of 1.3 kb and a 4.2 kb band (see A) in the two <i>Δp52</i> clones (<i>Δp52-1 and Δp52 -2</i>). (C) PCR analysis of genomic DNA of Wt and <i>Δp52</i> clones and the plasmid DNA (construct) demonstrates correct disruption of <i>p52</i>. Genomic DNA from Wt and <i>Δp52</i> asexual parasites and sporozoites was used as template for the PCR reactions. The Wt specific PCR was performed using primers 1638 and 1676 amplifying a 2.1 kb fragment. PCR primer pairs 1638 and L430, specific for integration of the DNA construct (see A) amplify a 2.0 kb fragment. Primer pairs 190 and 191 amplifying a 1.8 kb fragment from <i>T. gondii dhfr/ts</i> were used as a control.</p

Invasion capacity of Wt and Δ<i>p52</i> sporozoites in primary human hepatocytes <i>in vitro</i>.

<p>(A) Intra (In) and extracellular (Ex) sporozoites 3 hrs after incubation of sporozoites with primary human hepatocytes in culture. Sporozoites were first stained with anti-PfCSP antibodies (red). Then cells were permeabilised and sporozoites were stained with anti-PfCSP antibodies (green). Consequently, extracellular sporozoites will stain red AND green and intracellular sporozoites will stain only green. Nuclei of the hepatocytes (white arrow heads) were stained with DAPI (B) The percentage of intracellular/invaded sporozoites (Wt and Δ<i>p52</i> mutant lines) in primary human hepatocyte 3 hours after sporozoite incubation, as determined in the double anti-CSP staining immuno-fluorescence assay (see A). (C) The number of schizonts detected by IFA using anti-HSP70 antibodies and the nuclear dye DAPI formed 3 days after incubation with either Wt or Δ<i>p52</i> mutant sporozoites. (D) The number of schizonts detected by IFA using anti-HSP70 antibodies and the nuclear dye DAPI formed 5 days after incubation with either Wt or Δ<i>p52</i> mutant sporozoites.</p