Targeted disruption of py235ebp-1: Invasion of erythrocytes by Plasmodium yoelii using an alternative Py235 erythrocyte binding protein

Plasmodium yoelii YM asexual blood stage parasites express multiple members of the py235 gene family, part of the super-family of genes including those coding for Plasmodium vivax reticulocyte binding proteins and Plasmodium falciparum RH proteins. We previously identified a Py235 erythrocyte binding protein (Py235EBP-1, encoded by the PY01365 gene) that is recognized by protective mAb 25.77. Proteins recognized by a second protective mAb 25.37 have been identified by mass spectrometry and are encoded by two genes, PY01185 and PY05995/PY03534. We deleted the PY01365 gene and examined the phenotype. The expression of the members of the py235 family in both the WT and gene deletion parasites was measured by quantitative RT-PCR and RNA-Seq. py235ebp-1 expression was undetectable in the knockout parasite, but transcription of other members of the family was essentially unaffected. The knockout parasites continued to react with mAb 25.77; and the 25.77-binding proteins in these parasites were the PY01185 and PY05995/PY03534 products. The PY01185 product was also identified as erythrocyte binding. There was no clear change in erythrocyte invasion profile suggesting that the PY01185 gene product (designated PY235EBP-2) is able to fulfill the role of EBP-1 by serving as an invasion ligand although the molecular details of its interaction with erythrocytes have not been examined. The PY01365, PY01185, and PY05995/PY03534 genes are part of a distinct subset of the py235 family. In P. falciparum, the RH protein genes are under epigenetic control and expression correlates with binding to distinct erythrocyte receptors and specific invasion pathways, whereas in P. yoelii YM all the genes are expressed and deletion of one does not result in upregulation of another. We propose that simultaneous expression of multiple Py235 ligands enables invasion of a wide range of host erythrocytes even in the presence of antibodies to one or more of the proteins and that this functional redundancy at the protein level gives the parasite phenotypic plasticity in the absence of differences in gene expression

Passive Immunization with a Multicomponent Vaccine against Conserved Domains of Apical Membrane Antigen 1 and 235-Kilodalton Rhoptry Proteins Protects Mice against Plasmodium yoelii Blood-Stage Challenge Infection

During malaria parasite invasion of red blood cells, merozoite proteins bind receptors on the surface of the erythrocyte. Two candidate Plasmodium yoelii adhesion proteins are apical membrane antigen 1 (AMA1) and the 235-kDa rhoptry proteins (P235). Previously, we have demonstrated that passive immunization with monoclonal antibodies (MAbs) 45B1 and 25.77 against AMA1 and P235, respectively, protects against a lethal challenge infection with P. yoelii YM. We show that MAb 45B1 recognizes an epitope located on a conserved surface of PyAMA1, as determined by phage display and analysis of the three-dimensional structure of AMA1, in a region similar to that bound by the P. falciparum AMA1-specific inhibitory antibody 4G2. The epitope recognized by 25.77 could not be assigned. We report here that MAbs 45B1 and 25.77 also protect against challenge with the nonlethal parasite line 17X, in which PyAMA1 has a significantly different amino acid sequence from that in YM. When administered together, the two MAbs acted at least additively in providing protection against challenge with the virulent YM parasite. These results support the concept of developing a multicomponent blood-stage vaccine and the inclusion of polymorphic targets such as AMA1, which these results suggest contain conserved domains recognized by inhibitory antibodies

Systematic Genetic Analysis of the Plasmodium falciparum MSP7-Like Family Reveals Differences in Protein Expression, Location, and Importance in Asexual Growth of the Blood-Stage Parasite▿†‡

Proteins located on Plasmodium falciparum merozoites, the invasive form of the parasite's asexual blood stage, are of considerable interest in vaccine research. Merozoite surface protein 7 (MSP7) forms a complex with MSP1 and is encoded by a member of a multigene family located on chromosome 13. The family codes for MSP7 and five MSP7-related proteins (MSRPs). In the present study, we have investigated the expression and the effect of msrp gene deletion at the asexual blood stage. In addition to msp7, msrp2, msrp3, and msrp5 are transcribed, and mRNA was easily detected by hybridization analysis, whereas mRNA for msrp1 and msrp4 could be detected only by reverse transcription (RT)-PCR. Notwithstanding evidence of transcription, antibodies to recombinant MSRPs failed to detect specific proteins, except for antibodies to MSRP2. Sequential proteolytic cleavages of MSRP2 resulted in 28- and 25-kDa forms. However, MSRP2 was absent from merozoites; the 25-kDa MSRP2 protein (MSRP225) was soluble and secreted upon merozoite egress. The msrp genes were deleted by targeted disruption in the 3D7 line, leading to ablation of full-length transcripts. MSRP deletion mutants had no detectable phenotype, with growth and invasion characteristics comparable to those of the parental parasite; only the deletion of MSP7 led to a detectable growth phenotype. Thus, within this family some of the genes are transcribed at a significant level in asexual blood stages, but the corresponding proteins may or may not be detectable. Interactions of the expressed proteins with the merozoite also differ. These results highlight the potential for unexpected differences of protein expression levels within gene families

Novel antimalarial antibodies highlight the importance of the antibody Fc region in mediating protection

Parasite drug resistance and difficulties in developing effective vaccines have precipitated the search for alternative therapies for malaria. The success of passive immunization suggests that immunoglobulin (Ig)-based therapies are effective. To further explore the mechanism(s) by which antibody mediates its protective effect, we generated human chimeric IgG1 and IgA1 and a single-chain diabody specific for the C-terminal 19-kDa region of Plasmodium yoelii merozoite surface protein 1 (MSP119), a major target of protective immune responses. These novel human reagents triggered in vitro phagocytosis of merozoites but, unlike their parental mouse IgG2b, failed to protect against parasite challenge in vivo. Therefore, the Fc region appears critical for mediating protection in vivo, at least for this MSP119 epitope. Such antibodies may serve as prototype therapeutic agents, and as useful tools in the development of in vitro neutralization assays with Plasmodium parasites