ABSTRACT
BACKGROUND: Rodent malaria parasites (RMP) are used extensively as models of human malaria. Draft RMP genomes have been published for Plasmodium yoelii, P. berghei ANKA (PbA) and P. chabaudi AS (PcAS). Although availability of these genomes made a significant impact on recent malaria research, these genomes were highly fragmented and were annotated with little manual curation. The fragmented nature of the genomes has hampered genome wide analysis of Plasmodium gene regulation and function. RESULTS: We have greatly improved the genome assemblies of PbA and PcAS, newly sequenced the virulent parasite P. yoelii YM genome, sequenced additional RMP isolates/lines and have characterized genotypic diversity within RMP species. We have produced RNA-seq data and utilised it to improve gene-model prediction and to provide quantitative, genome-wide, data on gene expression. Comparison of the RMP genomes with the genome of the human malaria parasite P. falciparum and RNA-seq mapping permitted gene annotation at base-pair resolution. Full-length chromosomal annotation permitted a comprehensive classification of all subtelomeric multigene families including the 'Plasmodium interspersed repeat genes' (pir). Phylogenetic classification of the pir family, combined with pir expression patterns, indicates functional diversification within this family. CONCLUSIONS: Complete RMP genomes, RNA-seq and genotypic diversity data are excellent and important resources for gene-function and post-genomic analyses and to better interrogate Plasmodium biology. Genotypic diversity between P. chabaudi isolates makes this species an excellent parasite to study genotype-phenotype relationships. The improved classification of multigene families will enhance studies on the role of (variant) exported proteins in virulence and immune evasion/modulation.
Subject(s)
Gene Expression , Genome, Protozoan , Plasmodium falciparum/genetics , Plasmodium/classification , Base Sequence , Chromosome Mapping , Gene Expression Regulation , Genotype , Molecular Sequence Data , Multigene Family , Plasmodium/genetics , Plasmodium falciparum/classification , RNA, Protozoan/genetics , Sequence Analysis, RNA , Transcriptome/geneticsABSTRACT
Merozoite surface protein 1 (MSP1) has been identified as a target antigen for protective immune responses against asexual blood stage malaria, but effective vaccines based on MSP1 have not been developed so far. We have modified the sequence of Plasmodium yoelii MSP119 (the C-terminal region of the molecule) and examined the ability of the variant proteins to bind protective monoclonal antibodies and to induce protection by immunization. In parallel, we examined the structure of the protein and the consequences of the amino acid changes. Naturally occurring sequence polymorphisms reduced the binding of individual protective antibodies, indicating that they contribute to immune evasion, but immunization with these variant proteins still provided protective immunity. One variant that resulted in the localized distortion of a loop close to the N-terminus of MSP119 almost completely ablated protection by immunization, indicating the importance of this region of MSP119 as a target for protective immunity and in vaccine development.
Subject(s)
Antibodies, Protozoan/immunology , Merozoite Surface Protein 1/metabolism , Models, Molecular , Plasmodium yoelii/metabolism , Protozoan Proteins/metabolism , Amino Acid Substitution , Animals , Antibodies, Monoclonal/immunology , Escherichia coli/metabolism , Malaria Vaccines/immunology , Merozoite Surface Protein 1/chemistry , Merozoite Surface Protein 1/genetics , Mice , Mice, Inbred BALB C , Protein Structure, Tertiary , Protozoan Proteins/chemistry , Protozoan Proteins/genetics , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunologyABSTRACT
Apicomplexan parasites invade host cells by a conserved mechanism: parasite proteins are secreted from apical organelles, anchored in the host cell plasma membrane, and then interact with integral membrane proteins on the zoite surface to form the moving junction (MJ). The junction moves from the anterior to the posterior of the parasite resulting in parasite internalization into the host cell within a parasitophorous vacuole (PV). Conserved as well as coccidia-unique rhoptry neck proteins (RONs) have been described, some of which associate with the MJ. Here we report a novel RON, which we call RON12. RON12 is found only in Plasmodium and is highly conserved across the genus. RON12 lacks a membrane anchor and is a major soluble component of the nascent PV. The bulk of RON12 secretion happens late during invasion (after parasite internalization) allowing accumulation in the fully formed PV with a small proportion of RON12 also apparent occasionally in structures resembling the MJ. RON12, unlike most other RONs is not essential, but deletion of the gene does affect parasite proliferation. The data suggest that although the overall mechanism of invasion by Apicomplexan parasites is conserved, additional components depending on the parasite-host cell combination are required.
Subject(s)
Plasmodium falciparum/growth & development , Protozoan Proteins/metabolism , Endocytosis , Gene Deletion , Plasmodium falciparum/genetics , Protozoan Proteins/geneticsABSTRACT
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.
Subject(s)
Alternative Splicing , Antigens, Protozoan/genetics , Erythrocytes/parasitology , Gene Deletion , Malaria/genetics , Plasmodium yoelii/growth & development , Plasmodium yoelii/pathogenicity , Amino Acid Sequence , Animals , Antibodies, Monoclonal , Blotting, Southern , Blotting, Western , Erythrocyte Count , Erythrocytes/immunology , Erythrocytes/metabolism , Fluorescent Antibody Technique , Genome, Protozoan , Immunoprecipitation , Malaria/parasitology , Mice , Mice, Inbred BALB C , Mice, Knockout , Molecular Sequence Data , Multigene Family , Plasmodium yoelii/genetics , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction , Sequence Homology, Amino Acid , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
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 (MSRP2(25)) 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.
Subject(s)
Life Cycle Stages/genetics , Membrane Proteins/metabolism , Multigene Family , Plasmodium falciparum/growth & development , Plasmodium falciparum/genetics , Protozoan Proteins/metabolism , Reproduction, Asexual/genetics , Animals , Erythrocytes/parasitology , Gene Deletion , Gene Expression Regulation , Gene Knockout Techniques , Genes, Protozoan , Genetic Loci/genetics , Humans , Membrane Proteins/genetics , Protein Transport , Protozoan Proteins/genetics , Transcription, GeneticABSTRACT
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.
Subject(s)
Genes, Protozoan/genetics , Malaria/genetics , Plasmodium yoelii/genetics , Protozoan Proteins/genetics , Amino Acid Sequence , Animals , Base Sequence , Blotting, Western , Chromatography, High Pressure Liquid , Electrophoresis, Polyacrylamide Gel , Enzyme-Linked Immunosorbent Assay , Fluorescent Antibody Technique , Malaria/immunology , Malaria/metabolism , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Plasmodium yoelii/immunology , Protozoan Proteins/immunology , Protozoan Proteins/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Recombinant Proteins/metabolism , Sequence Homology, Amino AcidABSTRACT
Highly purified protein antigens are usually poor immunogens; in practice, adjuvants are needed to obtain satisfactory immune responses. Plasmodium yoelii 19-kDa merozoite surface protein 1 (MSP1(19)) is a weak antigen, but mice vaccinated with this antigen in strong adjuvants can survive an otherwise lethal parasite challenge. Fusion proteins comprising this antigen fused to the oligomerization domain of the murine complement inhibitor C4-binding protein (C4bp) and a series of homologues have been produced. These C4bp domains acted as adjuvants for the fused antigen; the MSP1(19)-murine C4bp fusion protein induced protective immunity in BALB/c mice. Because this fusion protein also induced antibodies against circulating murine C4bp, distantly related C4bp oligomerization domains fused to the same antigen were tested. These homologous domains did not induce antibodies against murine C4bp and, surprisingly, induced higher antibody titers against the antigen than the murine C4bp domain induced. These results demonstrate a new adjuvantlike effect of C4bp oligomerization domains.
Subject(s)
Adjuvants, Immunologic , Histocompatibility Antigens/immunology , Malaria/prevention & control , Merozoite Surface Protein 1/immunology , Recombinant Fusion Proteins/immunology , Amino Acid Sequence , Animals , Antibodies, Protozoan/blood , Enzyme-Linked Immunosorbent Assay , Fluorescent Antibody Technique, Indirect , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Parasitemia/prevention & control , Plasmodium yoelii/immunology , Protein Structure, Tertiary , Sequence AlignmentABSTRACT
In Plasmodium falciparum, merozoite surface protein 7 (MSP7) was originally identified as a 22kDa protein on the merozoite surface and associated with the MSP1 complex shed during erythrocyte invasion. MSP7 is synthesised in schizonts as a 351-amino acid precursor that undergoes proteolytic processing. During biosynthesis the MSP1 and MSP7 precursors form a complex that is targeted to the surface of developing merozoites. In the sequential proteolytic processing of MSP7, N- and C-terminal 20 and 33kDa products of primary processing, MSP7(20) and MSP7(33) are formed and MSP7(33) remains bound to full length MSP1. Later in the mature schizont, MSP7(20) disappears from the merozoite surface and on merozoite release MSP7(33) undergoes a secondary cleavage yielding the 22kDa MSP7(22) associated with MSP1. In free merozoites, both MSP7(22) and a further cleaved product, MSP7(19) present only in some parasite lines, were detected; these two derivatives are shed as part of the protein complex with MSP1 fragments during erythrocyte invasion. Primary processing of MSP7 is brefeldin A-sensitive while secondary processing is resistant to both calcium chelators and serine protease inhibitors. Primary processing of MSP7 occurs prior to that of MSP1 in a post-Golgi compartment, whereas the secondary cleavage occurs on the surface of the developing merozoite, possibly at the time of MSP1 primary processing and well before the secondary processing of MSP1.
Subject(s)
Erythrocytes , Membrane Proteins/metabolism , Peptide Hydrolases/metabolism , Plasmodium falciparum/physiology , Protein Biosynthesis/genetics , Protein Processing, Post-Translational , Protozoan Proteins/metabolism , Animals , Brefeldin A/pharmacology , Erythrocytes/metabolism , Membrane Proteins/genetics , Microscopy, Electron, Transmission , Microscopy, Immunoelectron , Plasmodium falciparum/ultrastructure , Protein Binding , Protein Processing, Post-Translational/drug effects , Protozoan Proteins/genetics , Schizonts/metabolism , Spectrometry, FluorescenceABSTRACT
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.
Subject(s)
Antibodies, Monoclonal/therapeutic use , Antigens, Protozoan/immunology , Immunization, Passive , Malaria Vaccines , Malaria/prevention & control , Membrane Proteins/immunology , Plasmodium yoelii/immunology , Protozoan Proteins/immunology , Amino Acid Sequence , Animals , Antibodies, Monoclonal/immunology , Antigens, Protozoan/chemistry , Blood/parasitology , Epitopes/chemistry , Epitopes/immunology , Membrane Proteins/chemistry , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Protein Structure, Tertiary , Protozoan Proteins/chemistrySubject(s)
Antibodies, Monoclonal/metabolism , Antigens, Protozoan/metabolism , Erythrocytes/parasitology , Plasmodium yoelii/pathogenicity , Protozoan Proteins/metabolism , Receptors, Cell Surface/metabolism , Amino Acid Sequence , Animals , Antibodies, Monoclonal/immunology , Antibodies, Protozoan/immunology , Antibodies, Protozoan/metabolism , Antigens, Protozoan/chemistry , Antigens, Protozoan/genetics , Molecular Sequence Data , Peptide Mapping , Plasmodium yoelii/genetics , Plasmodium yoelii/growth & development , Plasmodium yoelii/metabolism , Protozoan Proteins/chemistry , Protozoan Proteins/genetics , Receptors, Cell Surface/chemistry , Receptors, Cell Surface/geneticsABSTRACT
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 yoelii and Plasmodium falciparum, respectively. We have identified and disrupted msp7 in the Plasmodium berghei gene 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 msp7 was 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.
Subject(s)
Erythrocytes/parasitology , Membrane Proteins/genetics , Membrane Proteins/metabolism , Plasmodium berghei/growth & development , Plasmodium berghei/genetics , Protozoan Proteins/genetics , Protozoan Proteins/metabolism , Animals , Erythrocytes/cytology , Mice , Mice, Inbred BALB CABSTRACT
The 19 kDa C-terminal fragment of the malaria parasite merozoite surface protein 1 (MSP1(19)) is a leading malaria vaccine candidate. In rodents, high antibody levels to this protein confer protective immunity, and can be generated by immunization with the antigen in adjuvants. In natural human infections, however, MSP1(19)-specific antibody responses can be short-lived and comparatively low, despite repeated exposure to infection. The tightly folded structure of MSP1(19) is stabilized by five or six disulfide bonds. These bonds impede antigen processing and, thereby, may affect the generation of CD4+ T cells providing help for B cells. Asparagine endopeptidase could digest unfolded, but not native MSP1(19) in vitro. Immunization with unfolded MSP1(19) resulted in a faster antibody response, and a combination of unfolded and native MSP1(19) increased antibody responses to the native form. Immunization with either form of the antigen activated similar numbers of CD4+ T cells, but, unlike the antibody response, CD4+ T cells immunized with one form of MSP119 were able to respond in vitro to the other form of the protein. Although the reduced form of MSP1(19) does not induce protective antibodies, our data suggest that inclusion of unfolded protein may improve the efficacy of MSP1(19) as a vaccine.
Subject(s)
Antibodies, Protozoan/blood , Malaria Vaccines/chemistry , Malaria Vaccines/immunology , Malaria/prevention & control , Merozoite Surface Protein 1/chemistry , Merozoite Surface Protein 1/immunology , Amino Acid Sequence , Animals , Antigen Presentation , CD4-Positive T-Lymphocytes/immunology , Cysteine Endopeptidases/metabolism , Disulfides , Female , Lysosomes/enzymology , Malaria/immunology , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Plasmodium chabaudi/chemistry , Plasmodium yoelii/chemistry , Sequence AlignmentABSTRACT
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.
Subject(s)
Antibodies, Protozoan/immunology , Immunization, Passive , Immunoglobulin A/immunology , Immunoglobulin Fc Fragments/immunology , Immunoglobulin G/immunology , Merozoite Surface Protein 1/immunology , Plasmodium yoelii/immunology , Animals , Antibodies, Protozoan/chemistry , Enzyme Activation , Epitopes/immunology , Female , Humans , Immunoglobulin A/chemistry , Immunoglobulin G/chemistry , Malaria/prevention & control , Mice , Mice, Inbred BALB C , NADPH Oxidases/metabolism , Neutralization Tests , Neutrophils/enzymology , Neutrophils/immunology , Parasitemia/prevention & control , Phagocytosis , Recombinant Fusion Proteins/immunologyABSTRACT
Glycosylphosphatidyl-inositols (GPIs) are vital major glycoconjugates in intraerythrocytic stages of Plasmodium. Here, we report on the biosynthesis and the characterization of GPIs synthesized by the murine malarial parasite P. yoelii yoelii YM. Parasitized erythrocytes were labeled in vivo and in vitro with either radioactive nucleotide sugar precursors, ethanolamine or glucosamine. The pathway leading to the formation of GPI precursors was found to resemble that described for P. falciparum; however, in P. yoelii, the formation of an additional hydrophilic precursor containing an acid-labile modification was detected. The data suggest that this modification is linked to the fourth mannose attached to the trimannosyl backbone in an alpha1-2 linkage. The modification was susceptible to hydrofluoric acid (HF), but not to nitrous acid (HNO(2)). Data obtained from size-exclusion chromatography on Bio-Gel P4, and Mono Q analysis of the fragments generated by HNO(2) deamination suggest that the modification is due to the presence of an additional ethanolamine linked to the fourth mannose via a phosphodiester bond.
Subject(s)
Glycosylphosphatidylinositols/metabolism , Malaria/metabolism , Plasmodium yoelii , Animals , Carbohydrate Sequence , Chromatography, Ion Exchange , Chromatography, Thin Layer , Ethanolamines/metabolism , Female , Glycosylphosphatidylinositols/chemistry , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Molecular StructureABSTRACT
The 19 kDa carboxy-terminal domain of Plasmodium yoelii merozoite surface protein-1 (MSP1(19)) was expressed in Salmonella vaccine strains as a carboxy-terminal fusion to fragment C of tetanus toxin (TetC). This study demonstrates that antibodies that recognize disulphide-dependent conformational epitopes in native MSP1 react with the TetC-MSP1(19) fusion protein expressed in Salmonella. The proper folding of MSP1(19) polypeptide is dependent on both the Salmonella host strain and the protein to which the MSP1(19) polypeptide is fused. Serum from mice immunized with Salmonella typhimurium C5aroD expressing TetC-MSP1(19) recognized native MSP1 as shown by immunofluorescence with P. yoelii-infected erythrocytes. Antibody levels to MSP1(19) were highest in out-bred mice immunized with S. typhimurium C5aroD carrying pTECH2-MSP1(19) and antibody was mostly directed against reduction-sensitive conformational epitopes. However, antibody levels were lower than in BALB/c mice immunized with a glutathione S-transferase (GST)-MSP1(19) fusion protein in Freund's adjuvant, and which were protected against P. yoelii challenge infection. In challenge experiments with P. yoelii the Salmonella-immunized mice were not protected, probably reflecting the magnitude of the antibody response. The results of this study have important implications in the design of live multivalent bacterial vaccines against eukaryotic pathogens.