Molecular and functional characterization of vacuolar-ATPase from the American dog tick Dermacentor variabilis.

Vacuolar (V)-ATPase is a proton-translocating enzyme that acidifies cellular compartments for various functions such as receptor-mediated endocytosis, intracellular trafficking and protein degradation. Previous studies in Dermacentor variabilis chronically infected with Rickettsia montanensis have identified V-ATPase as one of the tick-derived molecules transcribed in response to rickettsial infection. To examine the role of the tick V-ATPase in tick-Rickettsia interactions, a full-length 2887-bp cDNA (2532-bp open reading frame) clone corresponding to the transcript of the V0 domain subunit a of D. variabilis V-ATPase (DvVATPaseV0a) gene encoding an 843 amino acid protein with an estimated molecular weight of ~96 kDa was isolated from D. variabilis. Amino acid sequence analysis of DvVATPaseV0a showed the highest similarity to VATPaseV0a from Ixodes scapularis. A potential N-glycosylation site and eight putative transmembrane segments were identified in the sequence. Western blot analysis of tick tissues probed with polyclonal antibody raised against recombinant DvVATPaseV0a revealed the expression of V-ATPase in the tick ovary. Transcriptional profiles of DvVATPaseV0a demonstrated a greater mRNA expression in the tick ovary, compared with the midgut and salivary glands; however, the mRNA level in each of these tick tissues remained unchanged after infection with R. montanensis for 1 h. V-ATPase inhibition bioassays resulted in a significant decrease in the ability of R. montanensis to invade tick cells in vitro, suggesting a role of V-ATPase in rickettsial infection of tick cells. Characterization of tick-derived molecules involved in rickettsial infection is essential for a thorough understanding of rickettsial transmission within tick populations and the ecology of tick-borne rickettsial diseases.

Rosetting in Plasmodium falciparum: a cytoadherence phenotype with multiple actors.

The capacity of Plasmodium falciparum-infected red blood cells to bind uninfected red blood cells ("rosetting") has been associated with high parasite density in numerous geographic areas and with severe malaria in African children. We summarize here the associations that have emerged from field studies and describe the various experimental models of rosetting that have been developed. A variety of erythrocyte receptors, several serum factors and a number of rosette-mediating PfEMP1 adhesins have been identified. Several var genes code for rosette-forming PfEMP1 adhesins in each P. falciparum genome, so that each clonal line has the capacity to generate distinct types of rosettes. To clarify their respective role in malaria pathogenesis, each of the multiple ligand/receptor interactions should be further studied for fine specificity, binding affinity and the impact of the large population polymorphism of the parasite variant repertoires should be assessed. Interestingly, some major human erythrocyte surface polymorphisms have been identified as affecting rosette formation, consistent with a role for rosetting in life-threatening falciparum malaria.

Human T-cell lymphotropic virus type 1 gag indeterminate western blot patterns in Central Africa: relationship to Plasmodium falciparum infection.

To gain insight on the significance of human T-cell lymphotropic virus type 1 (HTLV-1) indeterminate serological reactivities, we studied villagers of South Cameroon, focusing on a frequent and specific HTLV-1 Gag indeterminate profile (HGIP) pattern (gag p19, p26, p28, and p30 without p24 or Env gp21 and gp46). Among the 102 sera studied, 29 from all age groups had a stable HGIP pattern over a period of 4 years. There was no epidemiological evidence for sexual or vertical transmission of HGIP. Seventy-five percent of HGIP sera reacted positively on MT2 HTLV-1-infected cells by immunofluorescence assay. However, we could not isolate any HTLV-1 virus or detect the presence of p19 Gag protein in cultures of peripheral blood mononuclear cells obtained from individuals with strong HGIP reactivity. PCR experiments conducted with primers for HTLV-1 and HTLV-2 (HTLV-1/2 primers) encompassing different regions of the virus did not yield HTLV-1/2 proviral sequences from individuals with HGIP. Using 11 peptides corresponding to HTLV-1 or HTLV-2 immunodominant B epitopes in an enzyme-linked immunosorbent assay, one epitope corresponding to the Gag p19 carboxyl terminus was identified in 75% of HGIP sera, while it was recognized by only 41% of confirmed HTLV-1-positive sera. A positive correlation between HTLV-1 optical density values and titers of antibody to Plasmodium falciparum was also demonstrated. Finally, passage of sera through a P. falciparum-infected erythrocyte-coupled column was shown to specifically abrogate HGIP reactivity but not the HTLV-1 pattern, suggesting the existence of cross-reactivity between HTLV-1 Gag proteins and malaria-derived antigens. These data suggest that in Central Africa, this frequent and specific Western blot is not caused by HTLV-1 infection but could instead be associated with P. falciparum infection.

Evaluation of immunogenicity and protective efficacy of carrier-free Plasmodium falciparum R23 antigen in pre-exposed saimiri sciureus monkeys.

We have reported previously that the recombinant Glutathione S-transferase GTR23, induced protection after immunisation of naive or previously exposed Saimiri monkeys. We investigated the immunogenicity of carrier-free R23 repeats in pre-exposed animals in two adjuvant formulations. Three of five monkeys immunised with alum-formulated repeats and one of two animals immunised with the Polyalphaolefine formulation produced high titres of cytophilic antibodies with a primary type kinetics, indicating that the anti-P. falciparum antibodies present on the day of challenge were R23-specific. The four responders in R23-specific antibodies were protected against a challenge infection with the virulent FUP/SP strain. The other three animals failed to respond to immunisation and experienced an infection that required drug treatment. Unlike the other three animals that experienced an infection requiring drug treatment. These experiments support further development of the R23 repeats as a vaccine candidate.

A member of the Plasmodium falciparum Pf60 multigene family codes for a nuclear protein expressed by readthrough of an internal stop codon.

Four large multigene families have been described in Plasmodium falciparum malaria parasites (var, rif, stevor and Pf60). var and rif genes code for erythrocyte surface proteins and undergo clonal antigenic variation. We report here the characterization of the first Pf60 gene. The 6.1 gene is constitutively expressed by all mature blood stages and codes for a protein located within the nucleus. It has a single copy, 7-exon, 5' domain, separated by an internal stop codon from a 3' domain that presents a high homology with var exon II. Double-site immunoassay and P. falciparum transient transfection using the reporter luciferase gene demonstrated translation through the internal ochre codon. The 6.1 N-terminal domain has no homology with any protein described to date. Sequence analysis identified a leucine zipper and a putative nuclear localization signal and showed a high probability for coiled coils. Evidence for N-terminal coiled coil-mediated protein interactions was obtained. This identifies the 6.1 protein as a novel nuclear protein. These data show that the Pf60 and var genes form a superfamily with a common 3' domain, possibly involved in regulating homo- or heteromeric interactions.

Seasonal fluctuation of antibody levels to Plasmodium falciparum parasitized red blood cell-associated antigens in two Senegalese villages with different transmission conditions.

The recombinant R23, PfEB200, and GST-5 antigens derive from conserved antigens associated with the Plasmodium falciparum-infected erythrocyte membrane. They were identified as targets of protective antibodies in the Saimiri sciureus model. We have assessed here the humoral response to these antigens in humans. Cross-sectional surveys were conducted in two Senegalese villages with different levels of endemicity. The prevalence of specific IgG and IgM was similar and influenced by age in both localities. The anti-R23 antibodies decreased after the rainy season, particularly in the children less than ten years old. The anti-PfEB200 response did not show significant seasonal variation. The anti-GST-5 response increased in both the less-than 10-year-old and the greater-than 10-year-old groups after the rainy season in Dielmo, but only in the Ndiop villagers who were more than 10-years-old. Thus, antigen-specific seasonal variations of antibody levels were influenced differently by age in both villages. The isotype distribution was antigen-specific and differed for both seasons.

Novel target antigens of the variant-specific immune response to Plasmodium falciparum identified by differential screening of an expression library.

A primary infection by the Plasmodium falciparum Palo Alto O and R antigenic variants induces a variant-specific immunity in the Saimiri sciureus monkey. We have shown that these variants express distinct PfEMP1 antigens and differ in their levels of expression of additional antigens, including two conserved erythrocyte membrane-associated proteins, HRP1 and PfEMP3. To identify the antigens eliciting a variant-specific response, we conducted a differential screening of a lambdagt11 library with variant-specific sera. We report here the analysis of the 46 anti-R-specific clones. Two specific targets of the anti-R response were identified: (i) PfEMP3, suggesting that immunogenicity of this antigen is modulated by its relative abundance in different variants, and (ii) Asn-rich motifs. Most anti-R-specific clones, derived from so-far-undescribed genes, were detected by a cross-reaction on poly(Asn) stretches, as indicated by elimination of the signal after absorption on Asn-rich sequences. Reverse transcription-PCR (RT-PCR) showed that expression of the gene defined by clone 13 was R specific. Pepscan analysis of clone 13 identified three Asn-rich polypeptides and one unique peptide reacting specifically with antibodies eluted from the R-infected erythrocyte surface. Antisera raised to the unique peptide reacted with an R-specific protein. Attempts to demonstrate that clone 13 was derived from a var gene by using PCRs combining clone 13 and var-derived primers were unsuccessful. The var genes expressed by O and R parasites were identified not by this strategy but by RT-PCR with var-specific primers. This work has provided novel insights into immunity to antigenic variants and has identified a novel gene switched on during antigenic variation.

Evidence for distinct prototype sequences within the Plasmodium falciparum Pf60 multigene family.

Using oligonucleotides derived from Pf60.1, a member of the Plasmodium falciparum Pf60 multigene family, numerous fragments were amplified from genomic and cDNA from the 3D7 P. falciparum clone. DNA sequencing showed that the various fragments presented considerable diversity, indicating that the 3D7 repertoire contains at least 20 distinct versions of the region analysed. The various sequences aligned with either of two prototype sequences. Characteristic of the A-type was the presence of a 21 bp motif, present in variable copy number, as well as a sequence homologous to the Babesia sp. RAP-1 consensus. The B prototype sequence did not present such features and substantially differed from the A-type, due to accumulation of point mutations and numerous triplet deletions. Consistent with the marked differences between both sub-families, individual members from each sub-family did not cross-hybridise, produced distinct multiple band patterns on Southern blots and distinct chromosome profiles. Numerous hybrid sequences were observed. Interestingly, most var genes and var-related unspliced cDNAs described so far are of A/B hybrid type. These data suggest that the family has evolved by successive amplifications from two ancestral copies, with accumulation of mutations, as well as recombination and/or gene conversion events.

A large multigene family expressed during the erythrocytic schizogony of Plasmodium falciparum.

We report the identification of a large multigene family of Plasmodium falciparum using a clone isolated with a polyclonal antiserum raised to a Babesia divergens merozoite protein. The recombinant antigen reacted with human sera collected from individuals exposed to malaria. The deduced protein sequence contains a motif homologous to the consensus sequence of merozoite rhoptry proteins encoded by multigene families in several Babesia species. Antibodies raised to the recombinant protein reacted with a 60-kDa merozoite protein both on B. divergens and on P. falciparum immunoblots. The insert hybridized to a large number of fragments on P. falciparum Southern blots and to most chromosomes of the parasite. Specifically, approx. 3-kb RNAs were detected in 4-16-nucleus schizonts. Ten distinct cDNAs were isolated that differed in the size, position and number of restriction sites in the region homologous to the original genomic clone. With about 140 copies per haploid genome, this is the first large multigene family described in malaria parasites. The existence of a multigene family encoding proteins present in the invasive stage of malaria parasites suggests an important role in invasion and denotes a significant potential for generating diversity.

Immunogenicity and antigenicity of a Plasmodium falciparum protein fraction (90-110 kDa) able to protect squirrel monkeys against asexual blood stages.

A monkey vaccination trial using a Plasmodium falciparum protein fraction containing antigens of 90-110 kDa is reported. The fraction was obtained by electroelution from preparative polyacrylamide gels. Three monkeys out of five resisted a heavy challenge dose of highly virulent parasites. Using specific antisera, several components of the fraction were identified, namely heat shock protein 90 (hsp90), Ag44/RhopH3, ABRA, 96tR/GBP130 and Pf96 protease. The fraction did not contain KAHRP, nor the SERP antigen. The antibody response of the monkeys was studied on these individual antigens purified by preparative immunoprecipitation. Surprisingly, hsp90 was found in the immunoprecipitates obtained with SERP antisera. Interestingly, the response to hsp90 correlated with protection, high antibody titres being found only in the protected monkeys. In contrast, no correlation with protection could be found for the response to the other antigens.

Prediction of mortality and morbidity with a 6-minute walk test in patients with left ventricular dysfunction. SOLVD Investigators.

OBJECTIVE: To study the potential usefulness of the 6-minute walk test, a self-paced submaximal exercise test, as a prognostic indicator in patients with left ventricular dysfunction. DESIGN: Data were collected during a prospective cohort study, the Studies of Left Ventricular Dysfunction (SOLVD) Registry Substudy. SETTING: Twenty tertiary care hospitals in the United States, Canada, and Belgium. PARTICIPANTS: A stratified random sample of 898 patients from the SOLVD Registry who had either radiological evidence of congestive heart failure and/or an ejection fraction of 0.45 or less were enrolled in the substudy and underwent a detailed clinical evaluation including a 6-minute walk test. Patients were followed up for a mean of 242 days. OUTCOME MEASURES: Mortality and hospitalization. RESULTS: During follow-up, 52 walk-test participants (6.2%) died and 252 (30.3%) were hospitalized. Hospitalization for congestive heart failure occurred in 78 participants (9.4%), and the combined endpoint of death or hospitalization for congestive heart failure occurred in 114 walk-test participants (13.7%). Compared with the highest performance level, patients in the lowest performance level had a significantly greater chance of dying (10.23% vs 2.99%; P = .01), of being hospitalized (40.91% vs 19.90%; P = .002), and of being hospitalized for heart failure (22.16% vs 1.99%; P < .0001). In a logistic regression model, ejection fraction and distance walked were equally strong and independent predictors of mortality and heart failure hospitalization rates during follow-up. CONCLUSION: The 6-minute walk test is a safe and simple clinical tool that strongly and independently predicts morbidity and mortality in patients with left ventricular dysfunction.

Clinical characteristics of patients in studies of left ventricular dysfunction (SOLVD).

The Studies of Left Ventricular Dysfunction (SOLVD) trials were designed to evaluate the effects of enalapril on long-term mortality in patients with severe left ventricular (LV) dysfunction. Patients with LV ejection fractions less than or equal to 0.35 and symptoms of congestive heart failure (CHF) were enrolled in the treatment trial, whereas those with no history of overt CHF and taking no treatment directed for LV dysfunction were enrolled in the prevention trial. The baseline clinical characteristics of SOLVD patients were compared to characterize differences between patients in these 2 separate but concurrent trials. From over 70,000 patients screened with LV dysfunction, 4,228 patients were enrolled in the prevention trial and 2,569 patients in the treatment trial. Ischemic heart disease was the primary cause of LV dysfunction in both prevention (83%) and treatment (71%) trial patients. Prior myocardial infarction was present in 80% of the prevention and 66% of the treatment trial patients (p less than 0.001). In the prevention trial, infarction was recent (less than or equal to 6 months) in 27% patients and remote (greater than 6 months) in 57% patients. Treatment trial patients had proportionately more women (20 vs 13%; p less than 0.001) and non-Caucasians (20 vs 14%; p less than 0.001), as well as the coexisting risk factors of hypertension (42 vs 37%; p less than 0.001) and diabetes (26 vs 15%; p less than 0.001) than did prevention trial patients. Clinical characteristics of patients in both trials were influenced by the gender and race of enrolled patients. Similarly, coronary artery bypass surgery was performed less often in women and non-Caucasians.(ABSTRACT TRUNCATED AT 250 WORDS)

Plasmodium falciparum: characterization of gene R45 encoding a trophozoite antigen containing a central block of six amino acid repeats.

We describe here an antigen, called R45, expressed by the young trophozoites of Plasmodium falciparum. This antigen contains a block of tandem repeats of six amino acids which are recognized by sera from humans living in endemic areas. The R45 gene is located on chromosome 3. It is present in all strains examined and shows limited size polymorphism. The C-terminal unique region of the protein shows a strong homology with the catalytic domain of the serine protein kinases. Interestingly, the central repeats contain a large number of putative phosphorylation sites. The implications of these features are discussed.

Cross-reaction of antibodies to the nine-amino acid repeats of Plasmodium falciparum antigen 11.1 with human serum albumin.

Mice immunized with the recombinant antigen 11.1 beta-galactosidase, consisting of 22 repeats of the nine-amino acid unit from Plasmodium falciparum antigen 11.1, produced antibodies reacting with human serum albumin. A positive reaction was observed in dot-blot assays, in enzyme-linked immunosorbent assay and on immunoblots of sodium dodecyl sulfate polyacrylamide gels as well as two-dimensional gels. Binding was specific for human albumin, as no reaction could be detected on bovine serum albumin, hen egg ovalbumin, rat serum albumin or another abundant human serum protein, the alpha 2-macroglobulin. In addition, rabbit antibodies raised to human serum albumin reacted with keyhole lympet hemocyanin coupled to synthetic dimers of the nine-amino acid repeats of the P. falciparum 11.1 antigen. These data indicate antigenic relationship between the 11.1 antigen and human albumin. The proteins have a short sequence of homology in a region where human serum albumin differs from the albumins of other species.

Cross-reactive antigenic determinants present on different Plasmodium falciparum blood-stage antigens.

A gene encoding a previously undescribed antigen of Plasmodium falciparum has been isolated from a genomic expression library by use of a pool of human immune sera. Northern blot analysis indicated that the gene is expressed at the late stages of the intra-erythrocytic cycle. This antigen, 332, contains a series of degenerated amino acid repeats. Human antibodies affinity-purified on the 332 recombinant antigen reacted with a family of parasite proteins that are products of different genes. We identified antigens 11.1 and Pf155-RESA as members of this family and confirmed, using a human monoclonal antibody, the presence of cross-reacting determinants. The sequences of these antigens also share some structural homologies. The significance of this family of blood-stage antigens is discussed.

Plasmodium falciparum: molecular analysis of a putative protective antigen, the thermostable 96-kDa protein.

A group of three Plasmodium falciparum antigens of distinct pI, migrating with an apparent MW of 96 kDa has been previously identified as a target of protective immunity both in humans and in monkeys (Jouin et al. 1987, Dubois et al. 1987). These antigens are produced during the late stages of asexual intraerythrocytic development. One of these 96-kDa proteins, the 96 tR, has a pI of 5.25, is thermostable, and is released in the culture supernatant (Jouin et al. 1987). We report here the cloning and expression in Escherichia coli of the gene coding for this antigen. Antibodies raised to the recombinant 96 tR immunoprecipitated exclusively the 96 tR, indicating that the other two antigens of 96 kDa are the product(s) of distinct gene(s). Northern and Southern blots as well as DNA sequencing of the gene showed that the 96 tR antigen is identical to proteins identified in other laboratories as the glycophorin binding protein GBP 130 (Perkins 1984, Ravetch et al. 1985) and Ag 78 (Bianco et al. 1987). The 96-kDa antigen is produced at the trophozoite stage and more actively in the schizonts. It is released in the culture supernatant at the time of schizont rupture, together with two minor products, forming a characteristic triplet. This triplet was also detected in immunoblots of merozoites. An approximate quantification on immunoblots indicated that the largest proportion of the protein is found in the culture supernatant, a minor fraction being loosely associated with merozoites. By immunofluorescence and immunoelectron microscopy, intense signals were observed in the erythrocyte cytoplasm. The 50-amino acid repeats were found in all strains examined, the protein showing some size polymorphism. The antigen was detected in the serum of infected monkeys as well as in that of infected humans.

The S-antigen of Plasmodium falciparum Palo Alto represents a new S-antigen serotype.

The S-antigen from the Palo Alto isolate of Plasmodium falciparum has been characterized. The partial sequence for the gene coding for this antigen (clone 281) reveals the presence of tandem repeats of eight amino acids which defines a new S-antigen serotype. Antibodies raised against the 281 recombinant clone reacted with a 140 kDa antigen by immunoblotting with parasite extracts and culture supernatants. The 140 kDa peptide was also identified by immunoprecipitation of metabolic labelled parasites. The 281 mouse antiserum was used to localize the antigen on parasite smears by indirect immunofluorescence assay and more precisely by immunoelectron microscopy. The S-antigen is localized within the parasitophorous vacuole. Furthermore, different isolates were examined for the presence of the Palo Alto S-antigen specificity.

The polymorphic 11.1 locus of Plasmodium falciparum.

Clone pPF11.1 encodes a Plasmodium falciparum antigen expressed during the intraerythrocytic cycle and containing tandem repeats of a 9 amino acid unit. We report here an analysis of the genomic region specific for 11.1, which extends over 30 kb. It contains two blocks of repeats, spanning 13 kb and 9 kb. The restriction map suggests that the locus may result from a gene duplication. The 11.1 region is present in all P. falciparum strains examined so far. Southern analysis of 8 distinct isolates indicates that the locus is highly polymorphic. Thus the pPF11.1 repeats constitute a sensitive and discriminating probe to type P. falciparum strains.

Immunogenicity of Plasmodium falciparum antigenic determinants produced by Escherichia coli recombinant clones.

The immunogenicity of two parasite antigens produced by Escherichia coli as proteins fused to beta-galactosidase was investigated in three animal species: mice, rabbits and squirrel monkeys. 2L protein carries 71 amino acids of a parasite antigen and 11.1 protein carries 23 repeats of a 9-amino-acid repetitive unit. The humoral response was studied using indirect immunofluorescence and immunoprecipitation. The results indicate that immunization of mice, rabbits and squirrel monkeys using SDS-denatured 2L fusion protein induced antibodies able to bind to parasite antigen 2L in the IFA or in the immunoprecipitation assays. Immunization using the native fusion protein did not induce antibodies able to immunoprecipitate the 2L parasite antigen. The same observation was made for the animals immunized with 11.1 recombinant protein. In this case, the antibody response was also measured by ELISA using synthetic dimers of the repeat as antigen. In mice and rabbits, high titres of anti-11.1 antibodies were found by ELISA. However, when the antigen produced by the parasite itself was used to evaluate the response, low titres were found. This indicates that the animals produced high levels of antibodies to a structure which is not exposed in the parasite. In squirrel monkeys, the same observation was made, but the overall levels of the response to 11.1 antigen were considerably lower than those observed in mice or rabbits.