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ABSTRACTCircumsporozoite protein (CSP) of Plasmodium falciparumis a protective human malaria vaccine candidate. There is an urgent need for models that can rapidly down-select novel CSP-based vaccine candidates. In the present study, the mouse-mosquito transmission cycle of a transgenic Plasmodium bergheimalaria parasite stably expressing a functional full-length P. falciparumCSP was optimized to consistently produce infective sporozoites for protection studies. A minimal sporozoite challenge dose was established, and protection was defined as the absence of blood-stage parasites 14 days after intravenous challenge. The specificity of protection was confirmed by vaccinating mice with multiple CSP constructs of differing lengths and compositions. Constructs that induced high NANP repeat-specific antibody titers in enzyme-linked immunosorbent assays were protective, and the degree of protection was dependent on the antigen dose. There was a positive correlation between antibody avidity and protection. The antibodies in the protected mice recognized the native CSP on the parasites and showed sporozoite invasion inhibitory activity. Passive transfer of anti-CSP antibodies into naive mice also induced protection. Thus, we have demonstrated the utility of a mouse efficacy model to down-select human CSP-based vaccine formulations.

Plasmodiumparasites lacking plasmepsin 4 (PM4), an aspartic protease that functions in the lysosomal compartment and contributes to hemoglobin digestion, have only a modest decrease in the asexual blood-stage growth rate; however, PM4 deficiency in the rodent malaria parasite Plasmodium bergheiresults in significantly less virulence than that for the parental parasite. P. berghei Δpm4parasites failed to induce experimental cerebral malaria (ECM) in ECM-susceptible mice, and ECM-resistant mice were able to clear infections. Furthermore, after a single infection, all convalescent mice were protected against subsequent parasite challenge for at least 1 year. Real-time in vivoparasite imaging and splenectomy experiments demonstrated that protective immunity acted through antibody-mediated parasite clearance in the spleen. This work demonstrates, for the first time, that a single Plasmodiumgene disruption can generate virulence-attenuated parasites that do not induce cerebral complications and, moreover, are able to stimulate strong protective immunity against subsequent challenge with wild-type parasites. Parasite blood-stage attenuation should help identify protective immune responses against malaria, unravel parasite-derived factors involved in malarial pathologies, such as cerebral malaria, and potentially pave the way for blood-stage whole organism vaccines.

ABSTRACTDuring Toxoplasma gondiiinfection, a fraction of the multiplying parasites, the tachyzoites, converts into bradyzoites, a dormant stage, which form tissue cysts localized mainly in brain, heart, and skeletal muscles that persist for several years after infection. At this stage the parasite is protected from the immune system, and it is believed to be inaccessible to drugs. While the long persistence of tissue cysts does not represent a medical problem for healthy individuals, this condition represents a major risk for patients with a compromised immune system, who can develop recrudescent life-threatening T. gondiiinfections. We have investigated for the first time the dynamics and the kinetics of tachyzoite-to-bradyzoite interconversion and cyst formation in vivo by using stage-specific bioluminescent parasites in a mouse model. Our findings provide a new framework for understanding the process of bradyzoite differentiation in vivo. We have also demonstrated that complex molecules such as d-luciferin have access to tissue cysts and are metabolically processed, thus providing a rationale for developing drugs that attack the parasite at this developmental stage.

ABSTRACTThrombospondin-related adhesive protein of Cryptosporidium1 (TRAP-C1) belongs to a group of proteins that are also found in Toxoplasma gondii, Eimeria tenella, and Plasmodiumspecies. TRAP-related proteins are needed for gliding motility, host-cell attachment, and invasion. The objective of this study was to characterize the antibody response to recombinant TRAP-C1 (rTRAP-C1) in healthy volunteers exposed to C. parvumand their association with clinical illness. A total of 31 healthy adult volunteers participated. Seven volunteers received the C. parvumTAMU isolate (inocula, 10 to 300 oocysts), and 24 volunteers received the C. parvumUCP isolate (500 to 105oocysts). The total antibody (immunoglobulin M [IgM], IgG, and IgA) response to rTRAP C-1 was measured by enzyme-linked immunosorbent assays prior to and after exposure to Cryptosporidium parvum(days 0 to 45). Results of this study showed that individuals who were uninfected demonstrated higher reactivity at baseline compared to those who became infected. After challenge, increases in antibody reactivity were seen on days 30 and 45 compared to the results seen on days 0 to 5. The increases in antibody reactivity were statistically significant in subjects with diarrhea and with or without detectable oocysts compared to the results seen with those who were uninfected and asymptomatic. These findings suggest that increases in antibody reactivity to rTRAP-C1 occur after recent exposure to C. parvum.

The circumsporozoite protein of Plasmodium falciparumcontains two conserved motifs (regions I and II) that have been proposed to interact with mosquito and vertebrate host molecules in the process of sporozoite invasion of salivary glands and hepatocytes, respectively. To study the function of this protein we have replaced the endogenous circumsporozoite protein gene ofPlasmodium bergheiwith that of P. falciparumand with versions lacking either region I or region II. We show here that P. falciparumcircumsporozoite protein functions in rodent parasite and that P. bergheisporozoites carrying the P. falciparum CSgene develop normally, are motile, invade mosquito salivary glands, and infect the vertebrate host. Region I-deficient sporozoites showed no impairment of motility or infectivity in either vector or vertebrate host. Disruption of region II abolished sporozoite motility and dramatically impaired their ability to invade mosquito salivary glands and infect the vertebrate host. These data shed new light on the role of the CS protein in sporozoite motility and infectivity.

It is estimated that every year malaria infects ∼300 million people and accounts for the death of 2 million individuals. The Plasmodiumparasites that cause malaria in humans are transmitted exclusively by mosquito species belonging to the Anophelesgenus. The recent development of a gene transfer technology for Anopheles stephensimosquitoes, using the Minostransposable element marked with the enhanced green fluorescent protein EGFP (Catteruccia, F., Nolan, T., Loukeris, T. G., Blass, C., Savakis, C., Kafatos, F. C., and Crisanti, A. (2000) Nature405, 959–962), provides now a powerful tool to investigate the role of mosquito molecules involved in the interaction with the malaria parasite. Such technology, when further developed with additional markers and transposable elements, will be invaluable for analyzing the biology of the vector and for developing malaria-resistant mosquitoes to be used as a tool to control malaria transmission in the field. We report here the germline transformation of A. stephensimosquitoes using a piggyBac-based transposon to drive integration of the gene encoding for the red fluorescent protein dsRED.A. stephensiembryos were injected with transformation vector pPBRED containing the dsRED marker cloned within the arms of piggyBac. Microscopic analysis of G1larvae revealed the presence of seven fluorescent phenotypes whose different molecular origins were confirmed by Southern blotting analysis. Sequencing of the insertion sites in two lines demonstrated that integrations had occurred at TTAA nucleotides in accordance with piggyBac-mediated transpositions.

The Anopheles gambiaetrypsin family consists of seven genes that are transcribed in the gut of female mosquitoes in a temporal coordinated and mutually exclusive manner, suggesting the involvement of a complex transcription regulatory mechanism. We identified a highly conserved 12-nucleotide motif present in all A. gambiaeand Anopheles stephensitrypsin promoters. We investigated the role of this putative trypsin regulatory element (PTRE) in controlling the transcription of the trypsin genes. Gel shift experiments demonstrated that nuclear proteins of A. gambiaecell lines formed two distinct complexes with probes encompassing the PTRE sequence. Mapping of the binding sites revealed that one of the complex has the specificity of a GATA transcription factor. Promoter constructs containing mutations in the PTRE sequence that selectively abolished the binding of either one or both complexes exerted opposite effects on the transcriptional activity of trypsin promoters in A. gambiaeand Aedes aegypticell lines. In addition, the expression of a novel GATA gene was highly enriched in A. gambiaeguts. Taken together our data prove that factors binding to the PTRE region are key regulatory elements possibly involved in the blood meal-induced repression and activation of transcription in earlyand latetrypsin genes.

ABSTRACTThe micronemal protein 2 (MIC2) of Toxoplasma gondiishares sequence and structural similarities with a series of adhesive molecules of different apicomplexan parasites. These molecules accumulate, through a yet unknown mechanism, in secretory vesicles (micronemes), which together with tubular and membrane structures form the locomotion and invasion machinery of apicomplexan parasites. Our findings indicated that two conserved motifs placed within the cytoplasmic domain of MIC2 are both necessary and sufficient for targeting proteins to T. gondiimicronemes. The first motif is based around the amino acid sequence SYHYY. Database analysis revealed that a similar sequence is present in the cytoplasmic tail of all transmembrane micronemal proteins identified so far in different apicomplexan species. The second signal consists of a stretch of acidic residues, EIEYE. The creation of an artificial tail containing only the two motifs SYHYY and EIEYE in a preserved spacing configuration is sufficient to target the surface protein SAG1 to the micronemes ofT. gondii. These findings shed new light on the molecular mechanisms that control the formation of the microneme content and the functional relationship that links these organelles with the endoplasmic reticulum of the parasite.

We used contour‐clamped homogeneous electric field (CHEF) gel electrophoresis and Southern blot hybridization to analyze the molecular karyotype of Cryptosporidium parvumand establish the chromosomal location of 12 single copy genes. In agreement with previous studies, the molecular karyotype of C. parvumwas found to consist of partially co‐migrating chromosomes ranging in size from 0.97 to 1.55 Mb and segregating into five distinct electrophoretic bands. Hybridization results allowed the definition of a linkage group comprised of five distinct loci located on chromosome VI. Southern hybridization and restriction analysis of total C. parvumchromosomes or isolated chromosome VI using gene‐specific probes and an oligonucleotide specific for C. parvumtelomeres allowed the development of a long‐range restriction map of chromosome VI.

ABSTRACTToxoplasma gondiitachyzoites were transformed with the coding sequence of the circumsporozoite (CS) protein of the primate malaria parasite Plasmodium knowlesi. A single inoculation of live transformed tachyzoites elicited an antibody response directed against the immunodominant repeat epitope (EQPAAGAGG)2of the P. knowlesiCS protein in rhesus monkeys. Notably, these animals failed to show a positive serum conversion against T. gondii. Antibodies againstToxoplasmaantigens were detected only after a second inoculation with a higher number of transformed tachyzoites. This boost induced an increased antibody response against the P. knowlesiCS protein associated with immunoglobulin class switching, thus demonstrating the establishment of immunological memory. These results indicate that theToxoplasma-derived CS protein is efficiently recognized by the monkey immune system and represents an immunodominant antigen in transformed parasites.

Sporozoite recognition of host cells is a key step in the life-cycle of malaria parasites. Two sporozoite proteins have so far been characterized in some detail, the circumsporozoite protein (CS) and thrombospondin related adhesive protein (TRAP). We report here the cloning and expression of the TRAP gene homologue from Plasmodium berghei, PbTRAP. The PbTRAP gene encodes a protein of 606 amino acids having a deduced molecular mass of 66 kDa. The overall structure is clearly that of the TRAP family having a signal sequence followed by an integrin A domain, a sulphatide binding motif, followed by a proline based repeat before a transmembrane domain and helical cytoplasmic tail. The observed molecular mass is almost 50% larger than expected, this can be explained almost entirely by the abnormal behaviour in SDS-PAGE of the proline based repeat. As would be expected PbTRAP shows greatest similarity with the P. yoelii TRAP homologue sporozoite surface protein 2 (SSP2) than with PITRAP, the TRAP gene from P. falciparum. The pattern of expression is similar to that of SSP2.

Cryptosporidium wrairiwas isolated from guinea pigs during a spontaneous outbreak of cryptosporidiosis. Despite the morphological and antigenic similarities to C. parvum, C. wrairidisplayed a different host range and site of infection and may represent a separate species or sub‐species. We used the polymerase chain reaction to clone two distinct 550 bp‐long DNA fragments, Wc‐I and Wc‐II, of the gene encoding the Cryptosporidiumoocyst wall protein (COWP) of C. wrairi,which showed 98% identity to the C. parvumhomologue. Within Wc‐I, polymorphic RsaI restriction sites were used to develop a polymerase chain reaction‐restriction fragment length polymorphism method able to distinguish C. wrairifrom C. parvumand to identify two groups of C. parvumisolates differentially associated with animal and human infections.

ABSTRACTThe genetic analysis of oocysts recovered from the stools of humans and animals infected with Cryptosporidium parvumhas consistently shown the existence of two distinct genotypes. One of the genotypes is found exclusively in some human infections, whereas the other genotype is found in human as well as in animal infections. On the basis of these observations and the results of published epidemiological studies with single polymorphic markers, the existence of two separate transmission cycles has been postulated, one exclusively anthroponotic and the other involving both animals and humans. To test this hypothesis, C. parvumisolates of different geographic and host origins were analyzed by using unlinked genetic polymorphisms. A total of 28 isolates originating from Europe, North and South America, and Australia were examined. Isolates clustered into two groups, one comprising both human and animal isolates and the other comprising isolates only of human origin. The absence of recombinant genotypes is consistent with two reproductively isolated populations within the species C. parvum.

A Correction to this paper has been published: https://doi.org/10.1038/s41586-020-2956-7.

Merozoite invasion of red blood cells is crucial to the development of the parasite that causes malaria. Merozoite surface proteins (MSPs) mediate the first interaction between parasite and erythrocyte. In Plasmodium falciparum, they include a complex of products from at least 3 genes (msp1, msp6, and msp7), one of which, msp7, is part of a gene family containing 3 and 6 adjacent members in Plasmodium yoeliiand Plasmodium falciparum, respectively. We have identified and disrupted msp7in the Plasmodium bergheigene family. The protein is expressed in schizonts and colocalizes with MSP1. The synthesis and processing of MSP1 was unaffected in the parasite with the disrupted gene (MSP7ko). Disruption of msp7was not lethal but affected blood-stage parasite growth. MSP7ko parasites initially grew more slowly than wild-type parasites. However, when reticulocytes were prevalent, the rate of increase in parasitemia was similar, suggesting that MSP7ko parasites prefer to invade and grow within reticulocytes. (Blood. 2005;105:394-396)

We used contour-clamped homogeneous electric field (CHEF) gel electrophoresis and Southern blot hybridization to analyze the molecular karyotype of Cryptosporidium parvum and establish the chromosomal location of 12 single copy genes. In agreement with previous studies, the molecular karyotype of C. parvum was found to consist of partially co-migrating chromosomes ranging in size from 0.97 to 1.55 Mb and segregating into five distinct electrophoretic bands. Hybridization results allowed the definition of a linkage group comprised of five distinct loci located on chromosome VI. Southern hybridization and restriction analysis of total C. parvum chromosomes or isolated chromosome VI using gene-specific probes and an oligonucleotide specific for C. parvum telomeres allowed the development of a long-range restriction map of chromosome VI.