Cruzi Proteins Research Articles

Adhesion of Trypanosoma cruzi Trypomastigotes to Fibronectin or Laminin Modifies Tubulin and Paraflagellar Rod Protein Phosphorylation

BackgroundThe unicellular parasite Trypanosoma cruzi is the causative agent of Chagaś disease in humans. Adherence of the infective stage to elements of the extracellular matrix (ECM), as laminin and fibronectin, is an essential step in host cell invasion. Although members of the gp85/TS, as Tc85, were identified as laminin and fibronectin ligands, the signaling events triggered on the parasite upon binding to these molecules are largely unexplored.Methodology/Principal FindingsViable infective parasites were incubated with laminin, fibronectin or bovine serum albumin for different periods of time and the proteins were separated by bidimensional gels. The phosphoproteins were envisaged by specific staining and the spots showing phosphorylation levels significantly different from the control were excised and identified by MS/MS. The results of interest were confirmed by immunoblotting or immunoprecipitation and the localization of proteins in the parasite was determined by immunofluorescence. Using a host cell-free system, our data indicate that the phosphorylation contents of T. cruzi proteins encompassing different cellular functions are modified upon incubation of the parasite with fibronectin or laminin.Conclusions/SignificanceHerein it is shown, for the first time, that paraflagellar rod proteins and α-tubulin, major structural elements of the parasite cytoskeleton, are predominantly dephosphorylated during the process, probably involving the ERK1/2 pathway. It is well established that T. cruzi binds to ECM elements during the cell infection process. The fact that laminin and fibronectin induce predominantly dephosphorylation of the main cytoskeletal proteins of the parasite suggests a possible correlation between cytoskeletal modifications and the ability of the parasite to internalize into host cells.

Assignment of Putative Functions to Membrane “Hypothetical Proteins” from the Trypanosoma cruzi Genome

Protozoan parasites cause thousands of deaths each year in developing countries. The genome projects of these parasites opened a new era in the identification of therapeutic targets. However, the putative function could be predicted for fewer than half of the protein-coding genes. In this work, all Trypanosoma cruzi proteins containing predicted transmembrane spans were processed through an automated computational routine and further analyzed in order to assign the most probable function. The analysis consisted of dissecting the whole predicted protein in different regions. More than 5,000 sequences were processed, and the predicted biological functions were grouped into 19 categories according to the hits obtained after analysis. One focus of interest, due to the scarce information available on trypanosomatids, is the proteins involved in signal-transduction processes. In the present work, we identified 54 proteins belonging to this group, which were individually analyzed. The results show that by means of a simple pipeline it was possible to attribute probable functions to sequences annotated as coding for "hypothetical proteins." Also, we successfully identified the majority of candidates participating in the signal-transduction pathways in T. cruzi.

A microfluidic device based on a screen-printed carbon electrode with electrodeposited gold nanoparticles for the detection of IgG anti-Trypanosoma cruzi antibodies

In this article we report the development of an integrated microfluidic system coupled to a screen-printed carbon electrode (SPCE) applied to the quantitative determination of IgG specific antibodies present in serum samples of patients that suffer from Chagas disease. This relevant parasitic infection caused by the hemoflagellate protozoan Trypanosoma cruzi represents a major public health concern in Latin America. In order to perform the detection of mentioned antibodies, SPCE coupled to a microfluidic device was modified by electrodeposition of gold nanoparticles (AuNPs) and functionalized with Trypanosoma cruzi proteins from epimastigote membranes. The developed microfluidic immunosensor with immobilized T. cruzi proteins on the SPCE surface was successfully applied in the detection of specific IgG anti-T. cruzi antibodies, which were allowed to react immunologically with immobilized T. cruzi antigen. After that, labelled antibodies were quantified through the addition of horseradish peroxidase (HRP) enzyme-labeled secondary antibodies specific to human IgG, using 4-tert-butylcatechol (4-TBC) as enzymatic mediator. HRP in the presence of hydrogen peroxide (H(2)O(2)) catalyzes the oxidation of 4-TBC whose back electrochemical reduction was detected on a modified electrode at -100 mV. The calculated detection limit for electrochemical detection was 3.065 ng mL(-1) and the intra- and inter-assay coefficients of variation were below 6.95%.

The Trypanosoma cruzi proteins TcCox10 and TcCox15 catalyze the formation of heme A in the yeast Saccharomyces cerevisiae

Trypanosoma cruzi, the etiologic agent for Chagas’ disease, has requirements for several cofactors, one of which is heme. Because this organism is unable to synthesize heme, which serves as a prosthetic group for several heme proteins (including the respiratory chain complexes), it therefore must be acquired from the environment. Considering this deficiency, it is an open question as to how heme A, the essential cofactor for eukaryotic CcO enzymes, is acquired by this parasite. In the present work, we provide evidence for the presence and functionality of genes coding for heme O and heme A synthases, which catalyze the synthesis of heme O and its conversion into heme A, respectively. The functions of these T. cruzi proteins were evaluated using yeast complementation assays, and the mRNA levels of their respective genes were analyzed at the different T. cruzi life stages. It was observed that the amount of mRNA coding for these proteins changes during the parasite life cycle, suggesting that this variation could reflect different respiratory requirements in the different parasite life stages.

Subcellular Proteomics and Global Analysis of Posttranslational Modifications to Study Functional Roles of Trypanosoma cruzi Molecules

One century after the discovery of Chagas disease, the treatment for this illness is still based only on two drugs with limited efficacy and severe side effects. In this mini-review, we discuss the application of mass spectrometry (MS)- based proteomic approaches to study the biochemistry and cell biology of etiologic agent of Chagas disease, Trypanosoma cruzi. We focus the discussion in the analysis of subcellular proteomics and posttranslational modifications (PTMs). In re- cent years, subcellular proteomics has brought new insights into the localization of proteins and possible functions of or- ganelles. Thus far, proteomic analysis of reservosomes, ribosomes, detergent-solubilized membranes, and a preparation of an organelle mixture have been performed. In addition, a number of analyses of PTMs of T. cruzi proteins (i.e., histone modifications, phosphorylation, glycosylation, glycosylphosphatidylinositol (GPI)-anchoring, and nitrosylation) have been successfully carried out. The identification of those and other PTMs combined with cutting-edge biochemical, im- munological and cell biology approaches, have allowed a more in-depth understanding of biological and pathophysiologi- cal processes resulting from host cell-parasite interactions.

Biochemical studies with DNA polymerase β and DNA polymerase β-PAK of Trypanosoma cruzi suggest the involvement of these proteins in mitochondrial DNA maintenance

Mammalian DNA polymerase β is a nuclear enzyme involved in the base excision and single-stranded DNA break repair pathways. In trypanosomatids, this protein does not have a defined cellular localization, and its function is poorly understood. We characterized two Trypanosoma cruzi proteins homologous to mammalian DNA polymeraseβ, TcPolβ and TcPolβPAK, and showed that both enzymes localize to the parasite kinetoplast. In vitro assays with purified proteins showed that they have DNA polymerization and deoxyribose phosphate lyase activities. Optimal conditions for polymerization were different for each protein with respect to dNTP concentration and temperature, and TcPolβPAK, in comparison to TcPolβ, conducted DNA synthesis over a much broader pH range. TcPolβ was unable to carry out mismatch extension or DNA synthesis across 8-oxodG lesions, and was able to discriminate between dNTP and ddNTP. These specific abilities of TcPolβ were not observed for TcPolβPAK or other X family members, and are not due to a phenylalanine residue at position 395 in the C-terminal region of TcPolβ, as assessed by a site-directed mutagenesis experiment reversing this residue to a well conserved tyrosine. Our data suggest that both polymerases from T. cruzi could cooperate to maintain mitochondrial DNA integrity through their multiple roles in base excision repair, gap filling and translesion synthesis.

Proteomics in Trypanosoma cruzi – localization of novel proteins to various organelles

The completion of the genome sequence of Trypanosoma cruzi has been followed by several studies of protein expression, with the long-term aim to obtain a complete picture of the parasite proteome. We report a proteomic analysis of an organellar cell fraction from T. cruzi CL Brener epimastigotes. A total of 396 proteins were identified by LC-MS/MS. Of these, 138 were annotated as hypothetical in the genome databases and the rest could be assigned to several metabolic and biosynthetic pathways, transport, and structural functions. Comparative analysis with a whole cell proteome study resulted in the validation of the expression of 173 additional proteins. Of these, 38 proteins previously reported in other stages were not found in the only large-scale study of the total epimastigote stage proteome. A selected set of identified proteins was analyzed further to investigate gene copy number, sequence variation, transmembrane domains, and targeting signals. The genes were cloned and the proteins expressed with a c-myc epitope tag in T. cruzi epimastigotes. Immunofluorescence microscopy revealed the localization of these proteins in different cellular compartments such as ER, acidocalcisome, mitochondrion, and putative cytoplasmic transport or delivery vesicles. The results demonstrate that the use of enriched subcellular fractions allows the detection of T. cruzi proteins that are undetected by whole cell proteomic methods.

In silico reconstruction of the amino acid metabolic pathways of Trypanosoma cruzi

Trypanosoma cruzi is the epidemiological agent of Chagas' disease, affecting most of Central and South America, constituting a significant health and socio-economic problem. The parasite has a metabolism largely based on the consumption of amino acids, which participate in a diversity of metabolic pathways, leading to many crucial compounds for the survival of this parasite. Study of its enzymes has the potential to disclose new therapeutic targets and foster the development of new drugs. In this study, we employed computational approaches to reconstruct in silico the amino acid metabolic pathways of T. cruzi, aiming to link genomic information with functional information. For that, protein sequences from 570 EC classes belonging to 25 different pathways in general amino acid metabolism were downloaded from KEGG. A subset of 471 EC classes had at least one sequence deposited. Clustering of the proteins belonging to each EC class was performed using a similarity-based approach implemented in the tool AnEnPi. Reconstruction of the metabolic pathways comprising the amino acid metabolism of T. cruzi was performed by analyzing the output of BLASTP, using as query the dataset of predicted proteins of T. cruzi against all sequences of each individual cluster. This approach allowed us to identify 764 T. cruzi proteins probably involved in the metabolism of amino acids as well as the identification of several putative cases of analogy. Furthermore, we were able to identify several enzymatic activities of T. cruzi that were not previously included in KEGG.

Trypanosoma rangeli infected mouse sera reactivity with Trypanosoma cruzi synthetic peptides

In man, differential diagnosis of Trypanosoma cruzi and Trypanosoma rangeli infections represents a serious problem, not only because both parasites present similar geographical distribution, the same hosts and sometimes the same insect vector, but also because they have common antigen determinants. In this work IgM and IgG humoral responses to T. cruzi syntethic peptides in mice infected with T. cruzi and with T. rangeli were analysed. In a immunoradiometric assay ( IRMA ) 6 syntethic peptides were used, denominated as clones 1, 2, SAPA, 13, 30 and 36. The results showed that sera from infected mice with T. rangeli recognized all peptides derived from T. cruzi proteins, at IgM as well as IgG level. Reactivity with peptide SAPA is discussed as previous work indicated that SAPA is not codified in the T. rangeli genome. Our results support the suggestion that crossed reactions are due to the fact that both parasites present common antigens.

Cloning and expression analysis of two novel paraflagellar rod domain genes found in Trypanosoma cruzi

The eukaryotic flagellum is one of the most complex macromolecular structures found in cells, containing more than 250 proteins. One unique structure in the flagella of trypanomastids is the paraflagellar rod (PFR). The PFR constitutes a lattice of cytoskeletal filaments that lies alongside the axoneme in the flagella. This unique and complex structure is critical for cell motility, though little is known about its molecular assembly or its role in the lifecycle of trypanosomatids. These proteins are of particular importance in Trypanosoma cruzi, as purified or recombinant PFR proteins have been demonstrated to be immunogenic, protecting mice from a lethal challenge with the parasite. We have searched the T. cruzi databases and discovered two novel genes containing PFR domains. Both these genes are transcribed in vivo and are significantly larger than the previously described PFR genes identified in T. cruzi (>2 Kb). Real-time PCR was used to examine the relative expression levels of six PFR genes, including the two we describe here, in all three stages of T. cruzi's lifecycle. Database searches have further provided EST and genomic sequence support for the presence of these genes in two other pathogenic trypanosomatids, Trypanosoma brucei and Leishmania spp. One of these genes, designated PFR5 contains a carboxy terminal SH3 domain not previously seen in PFR family genes. We propose that this proline-binding SH3 domain may play an important role in the assembly of the PFR.

Identification of multiple HLA-A*0201-restricted cruzipain and FL-160 CD8 + epitopes recognized by T cells from chronically Trypanosoma cruzi-infected patients

Chronic Chagas disease occurs in 16 million individuals chronically infected by the protozoan Trypanosoma cruzi in Latin America, and may lead to a dilated cardiomyopathy in 10–30% of patients. A vigorous cellular immune response holds parasitism in check. However, up to now, few T. cruzi proteins have been shown to be recognized by CD8 + T cells from Chagas disease patients. In this study, we designed 94 peptides derived from T. cruzi proteins cruzipain and FL-160, predicted to bind to HLA-A2 molcules. After in vitro binding assays to HLA-A*0201, 26 peptides were selected, and their recognition by PBMC from Chagas disease patients was tested with the IFN-gamma ELISPOT assay. All 26 peptides were recognized by PBMC from at least one patient. Furthermore, a tetrameric HLA-A*0201 complex built with the cruzipain 60–68 peptide that was frequently recognized in the periphery also bound to CD8 + T cells from a heart-infiltrating T cell line obtained from a single patient with Chagas disease cardiomyopathy. Thus, our results suggest that the recognition of CD8 + T cell epitopes in cruzipain and FL-160 may have a pathogenic or protective role in chronic Chagas disease.

Immunosensor for the diagnosis of Chagas’ disease

Trypanosoma cruzi proteins from epimastigote membranes, herein referred as antigens, have been used for the construction of an amperometric immunosensor for serological diagnosis of Chagas' disease. The proteins used had a molecular mass ranging from 30 to 100 kDa. The gold electrode was treated with cysteamine and glutaraldehyde prior to antigen immobilization. Antibodies present in the serum of patients with Chagas' disease were captured by the immobilized antigens and the affinity interaction was monitored by chronoamperometry at a potential of -400 mV (versus Ag pseudo-reference electrode) using peroxidase-labeled IgG conjugate and hydrogen peroxide, iodide substrate. The incubation time to allow maximum antigen-antibody and antibody-peroxidase-labeled IgG interactions was 20 min with a reactivity threshold at -0.104 microA.

A cardiac myosin-specific autoimmune response is induced by immunization with Trypanosoma cruzi proteins.

Trypanosoma cruzi is the protozoan parasite that causes Chagas' heart disease, a potentially fatal cardiomyopathy prevalent in Central and South America. Infection with T. cruzi induces cardiac myosin autoimmunity in susceptible humans and mice, and this autoimmunity has been suggested to contribute to cardiac inflammation. To address how T. cruzi induces cardiac myosin autoimmunity, we investigated whether immunity to T. cruzi antigens could induce cardiac myosin-specific autoimmunity in the absence of live parasites. We immunized A/J mice with a T. cruzi Brazil-derived protein extract emulsified in complete Freund's adjuvant and found that these mice developed cardiac myosin-specific delayed-type hypersensitivity (DTH) and autoantibodies in the absence of detectable cardiac damage. The induction of autoimmunity was specific since immunization with extracts of the related protozoan parasite Leishmania amazonensis did not induce myosin autoimmunity. The immunogenetic makeup of the host was important for this response, since C57BL/6 mice did not develop cardiac myosin DTH upon immunization with T. cruzi extract. Perhaps more interesting, mice immunized with cardiac myosin developed T. cruzi-specific DTH and antibodies. This DTH was also antigen specific, since immunization with skeletal myosin and myoglobin did not induce T. cruzi-specific immunity. These results suggest that immunization with cardiac myosin or T. cruzi antigen can induce specific, bidirectionally cross-reactive immune responses in the absence of detectable cardiac damage.

Genetic immunization with LYT1 or a pool of trans-sialidase genes protects mice from lethal Trypanosoma cruzi infection

Genetic immunization with a limited set of genes has been demonstrated to be an effective means of protecting mice from a normally lethal challenge of Trypanosoma cruzi. The goal of this study was to expand the diversity of genes assessed as genetic vaccine candidates. Screening a T. cruzi amastigote cDNA expression library with anti-amastigote monoclonal antibodies resulted in the identification of two genes, the previously identified flagellar Ca 2+ binding protein, FCaBP, and a novel homologue of the adaptin AP-3 complex β3 subunit, Tcβ3. A third gene, LYT1, recently identified as a secreted T. cruzi protein involved in cell lysis and infectivity, and was selected. Although peptides from all three genes were found to be targets of cytotoxic T cell responses in chronically infected mice, only immunization with LYT1 protected mice from a normally lethal challenge of T. cruzi. As an alternative to testing individual T. cruzi genes as vaccines, pools of genes from the trans-sialidase (TS) and mucin families were assessed in vaccination studies. Immunization with pools of TS but not mucin genes provided protection against a normally lethal challenge of T. cruzi. This study demonstrates that the ability of T. cruzi proteins to elicit immune responses in infected hosts does not necessarily associate with the ability to induce protection and that both the products of single genes and multi-gene families may serve as effective vaccines.

Chagas disease: recombinant Trypanosoma cruzi antigens for serological diagnosis.

Diagnosis of individuals infected by Trypanosoma cruzi is performed mainly by serological tests using crude antigens, which might crossreact with other infections. In the past ten years, many recombinant T. cruzi proteins and synthetic peptides have been described, and some are already on the market. Managers of laboratories and blood banks need to make decisions on a cost-benefit basis whether to include these new-generation tests. Here, we indicate antigens that are likely to prove most useful.

Trypanosoma cruzi: A Putative Vacuolar ATP Synthase Subunit and a CAAX Prenyl Protease-Encoding Gene, as Examples of Gene Identification in Genome Projects

Porcel, B. M., Åslund, L., Pettersson, U., and Andersson B. 2000 Trypanosoma cruzi: A putative vacuolar ATP synthase subunit and a CAAX prenyl protease-encoding gene, as examples of gene identification in genome projects. Experimental Parasitology95, 176–186. An international genome program has been initiated to increase the knowledge about the Trypanosoma cruzi genome and thereby find effective tools to treat Chagas' disease. We here report the molecular characterization of two novel genes found in the course of this project. Two of the open reading frames (ORF) identified in the sequencing of the third smallest chromosome of the CL Brener strain of T. cruzi were selected for further molecular characterization due to their similarity to genes with interesting functions in other organisms and their potential as targets to combat the parasite. The first ORF (402 bp) showed homology to a 14-kDa vacuolar ATP synthase subunit F from a variety of organisms, such as yeast, rat, bovine, human, and a number of prokaryotes. The second ORF (1188 bp) resembled a CAAX prenyl protease-encoding gene, identified in different organisms, including Homo sapiens, Saccharomyces cerevisiae, and Arabidopsis thaliana, as well as several prokaryotes. RT-PCR from T. cruzi total epimastigote RNA allowed us to isolate the complete transcripts of these genes. Furthermore, screening of an available normalized cDNA library derived from the same stage of the parasite confirmed that both genes are expressed at least in the epimastigote stage of T. cruzi. Comparison of the putative T. cruzi proteins to their counterparts in other organisms revealed significant protein sequence conservation over large evolutionary distances. Computer analysis revealed the presence of several motifs in both proteins, possibly related to the regulation and localization of these proteins in the parasite.

A new liquid chromatography/tandem mass spectrometric approach for the identification of class I major histocompatibility complex associated peptides that eliminates the need for bioassays.

Cell-surface class I major histocompatibility complex (MHC) molecules present processed self- and nonself-peptides to thymus-derived (T) lymphocytes, allowing the intracellular compartment of cells to be sampled in order to detect infection. Since the class I MHC-peptide complex plays a critical role in cell-mediated immunity, it is important to obtain sequence information on the MHC-associated peptides unique to infected cells as a prelude to the development of vaccines. Here, we outline and test an alternative strategy for identifying the proteins that are processed through the MHC pathway. This new strategy eliminates the necessity of developing and maintaining cytotoxic T lymphocyte (CTL) lines for peptide identification. In this new approach genome sequences from the infecting agent are scanned for stretches of amino acids that match a particular MHC binding motif. Molecular masses from these putative MHC-binding peptide sequences are calculated and compared to those found for peptides isolated from pathogen-infected host cells using liquid chromatography/mass spectrometry (LC/MS). Peptides with masses matching those in the database are then analyzed by tandem mass spectrometry (MS/MS) to determine their identity. Using this approach we were able to confirm the processing and presentation of two Trypanosoma cruzi proteins by the MHC class I pathway. These data suggest that a rigorous approach employing two-dimensional separations in conjunction with MS/MS and bioinformatics is a feasible means of identifying pathogen gene products of immunological interest when a CTL assay is unavailable or unsuccessful.

Trypanosoma cruzi: Monoclonal Antibody to Cytoskeleton Recognizes Giant Proteins of the Flagellar Attachment Zone

Cytoskeletal preparations of Trypanosoma cruzi trypomastigotes and epimastigotes contain a protein recognized by a monoclonal antibody (2G4) which is connected to the flagellar attachment zone of both stages of the parasite. Western blot analysis revealed that the antibody was able to recognize protein bands of molecular masses higher than 700 kDa up to 2500 kDa. These giant proteins do not seem to share sequences with β-connectin since an anti-β-connectin antibody did not recognize the T. cruzi proteins nor did the 2G4 monoclonal antibody recognize authentic β-connectin. Immunofluorescence and immunogold electron microscopy provided evidence that this protein is located inside the cell body of the parasite, closely related to a corset of four microtubules known as subpellicular microtubule quartet. Immunogold labeling shows that the protein accompanies the flagellar attachment zone as long as the flagellum adheres to the cell body. It is proposed that these microtubule-associated proteins recognized by the 2G4 monoclonal antibody exist only in trypanosomatid forms having a junctional complex between the flagellum and the cell body and may act as transmembrane elements connecting the subpellicular microtubular quartet with the flagellum at the desmosome region.

Molecular and immunological characterization of a Trypanosoma cruzi protein homologous to mammalian elongation factor 1 gamma

In previous studies, we reported the characterization of three Trypanosoma cruzi proteins with molecular masses of 45, 30 and 25 kDa eluted from a glutathione agarose column (these proteins were named TcGBP). Using antibodies against TcGBP native proteins we could isolate from a lambda ZAPII epimastigote cDNA library cDNA clones encoding the 30 and 25 kDa proteins. Comparison of the two sequences with amino acid sequences in several data banks revealed that both protein sequences were highly homologous to human and Artemia salina elongation factor 1β. Thus, the proteins were named TcEF-1 β25 and TcEF-1 β30. In the present study we used a double immunoscreening strategy that allowed us to isolate a cDNA clone corresponding to the 45 kDa protein. The protein sequence revealed 31% identity and 61% homology with human and Artemia salina EF1γ and therefore was named TcEF-1γ. Moreover, three putative phosphorylation sites at position 51 (CSPC), at position 90 (RTPL) and at position 265 (PSPF) were found in the TcEF-1γ sequence. These sites are compatible with the notion that TcEF-1γ could be the target of phosphorylation by protein kinase(s). Random primed cDNA hybridized with a single 1.4 kb mRNA found in epimastigote, trypomastigote and amastigote forms. In addition, Southern blot analysis of genomic DNA suggested that the protein is encoded by a single gene. The TcEF-1γ cDNA was subcloned into the pGEX-4T-3 vector for expression in Escherichia coli. Antibodies against the fusion peptide allowed us to identify the weight sizes of the native protein (48 kDa) and its major degradation product (24.4 kDa) which are in close agreement with those of EF1-γ from Artemia salina and Schizosaccharomyces pombe. These antibodies reacted against macrophage cell line J774 extracts which indicates that EF-1γ epitopes were conserved throughout evolution.

BloodstreamTrypanosoma cruziparasites from mice simultaneously express antigens that are markers of acute and chronic human Chagas disease

Several recombinant Trypanosoma cruzi proteins previously isolated were used as antigens to analyse antibody specificities present in sera from human infections. Some parasite proteins such as SAPA (Shed Acute Phase Antigen) are antigenic early after infection. Others, like antigens 1 and 30, are antigenic mainly during the chronic phase of the infection. To understand why different proteins are antigenic at different periods of infection, specificities of antibodies present in the sera of infected mice were compared with the antigens expressed by parasites collected directly from blood. Parasites collected during the acute parasitaemia peak expressed not only antigen SAPA, but also antigens 1 and 30. However, only antibodies against SAPA were frequently observed during the early period and also in the chronic phase of murine infection. Long-lasting antibodies against SAPA were detected regardless of the mouse and parasite strains used. Furthermore, all 8 recombinant clones detected in a T. cruzi expression library with pooled sera from acutely infected mice were homologous to the SAPA gene. These results show that even though parasites from the acute parasitaemia peak in mice may express simultaneously several proteins known to be antigenic, only antibodies against SAPA were consistently detected.