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Previously, we identified a Plasmodium yoelii YM 140-kDa merozoite protein, designated PyP140, which formed a complex with apical membrane antigen 1 (AMA1). Furthermore, we produced a nonprotective monoclonal antibody (MAb), 48F8, that immunoprecipitated metabolically labeled PyP140 and localized the protein to the merozoite's apical end and, less frequently, to the merozoite surface, as observed by immunofluorescence assay (IFA). Here, using MAb 48F8, we have identified the pyp140 gene by screening a P. yoelii lambda-Zap cDNA expression library. The pyp140 cDNA covers approximately 90% of the putative open reading frame (ORF) of PY02159 from the P. yoelii NL genome sequencing project. Analysis of the complete gene identified the presence of two introns. The ORF encodes a 102,407-Da protein with an amino-terminal signal sequence, a series of three unique types of repeats, and a cysteine-rich region. The binding site of MAb 48F8 was also identified. A BLAST search with the deduced amino acid sequence shows significant similarity with the Toxoplasma gondii RON4 protein and the Plasmodium falciparum RON4 protein, and the sequence is highly conserved in other Plasmodium species. We produced the cysteine-rich domain of PyP140/RON4 by using the Pichia pastoris expression system and characterized the recombinant protein biochemically and biophysically. BALB/c mice immunized with the protein formulated in oil-in-water adjuvants produced antibodies that recognize parasitized erythrocytes by IFA and native PyP140/RON4 by immunoblotting but failed to protect against a lethal P. yoelii YM infection. Our results show that PyP140/RON4 is located within the rhoptries or micronemes. It may associate in part with AMA1, but the conserved cysteine-rich domain does not appear to elicit inhibitory antibodies, a finding that is supported by the marked sequence conservation in this protein within Plasmodium spp., suggesting that it is not under immune pressure.

The high molecular mass protein complex (RhopH) in the rhoptries of the malaria parasite consists of three distinct polypeptides with estimated sizes in Plasmodium falciparum of 155kDa (PfRhopH1), 140kDa (PfRhopH2) and 110kDa (PfRhopH3). Using a number of reagents, including a new mAb 4E10 that is specific for the PfRhopH complex, it was shown that the RhopH complex is synthesised during schizogony and transferred intact to the ring stage in newly invaded erythrocytes. The genes encoding RhopH1 and RhopH3 have already been identified and characterised in both P. falciparum and Plasmodium yoelii. In this report, we describe the identification of the gene for RhopH2 in both these parasite species. Peptide sequences were obtained from purified RhopH2 proteins and used to generate oligonucleotide primers and search malaria sequence databases. In a parallel approach, mAb 4E10 was used to identify a clone coding for RhopH2 from a P. falciparum cDNA library. The sequences of both P. falciparum and P. yoelii genes for RhopH2 were completed and compared. They both contain nine introns and there is a high degree of similarity between the deduced amino acid sequences of the two proteins. The P. falciparum gene is a single copy gene located on chromosome 9, and is transcribed in schizonts.

Vaccination of mice with the 42-kDa region of Plasmodium yoelii merozoite surface protein 1 (MSP1(42)) or its 19-kDa C-terminal processing product (MSP1(19)) can elicit protective antibody responses in mice. To investigate if the 33-kDa N-terminal fragment (MSP1(33)) of MSP1(42) also induces protection, the gene segment encoding MSP1(33) was expressed as a glutathione S-transferase (GST) fusion protein. C57BL/6 and BALB/c mice were immunized with GST-MSP1(33) and subsequently challenged with the lethal P. yoelii YM blood stage parasite. GST-MSP1(33) failed to induce protection, and all mice developed patent parasitemia at a level similar to that in naive or control (GST-immunized) mice; mice immunized with GST-MSP1(19) were protected, as has been shown previously. Specific prechallenge immunoglobulin G (IgG) antibody responses to MSP1 were analyzed by enzyme-linked immunosorbent assay and immunofluorescence. Despite being unprotected, several mice immunized with MSP1(33) had antibody titers (of all IgG subclasses) that were comparable to or higher than those in mice that were protected following immunization with MSP1(19). The finding that P. yoelii MSP1(33) elicits strong but nonprotective antibody responses may have implications for the design of vaccines for humans based on Plasmodium falciparum or Plasmodium vivax MSP1(42).