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In an effort to broaden the immune response induced by the RTS,S/AS02(A),vaccine, we have evaluated the immunogenicity of the RTS,S antigen when combined with MSP1(42) and with AMA1, antigens derived from the asexual blood stage. The objectives of this study were (i) to determine whether MSP1(42) and AMA1 vaccines formulated with the AS02(A) Adjuvant System were safe and immunogenic in the rhesus monkey model; (ii) to investigate whether MSP1(42) or AMA1 induced immune interference to each other, or to RTS,S, when added singly or in combinations at a single injection site; (iii) in the event of immune interference, to determine if this could be reduced when antigens were administered at separate sites. We found that MSP1(42) and AMA1 were safe and immunogenic, eliciting antibodies, and Th1 and Th2 responses using IFN-gamma and IL-5 as markers. When malaria antigens were delivered together in one formulation, MSP1(42) and RTS,S reduced AMA1-specific antibody responses as measured by ELISA however, only MSP1(42) lowered parasite growth inhibitory activity of anti-AMA1 antibodies as measured by in vitro growth inhibition assay. Unlike RTS,S, MSP1(42) significantly reduced AMA1 IFN-gamma and IL-5 responses. MSP1(42) suppression of AMA1 IFN-gamma responses was not seen in animals receiving RTS,S+AMA1+MSP1(42) suggesting that RTS,S restored IFN-gamma responses. Conversely, AMA1 had no effect on MSP1(42) antibody and IFN-gamma and IL-5 responses. Neither AMA1 alone or combined with MSP1(42) affected RTS,S antibody or IFN-gamma and IL-5 responses. Immune interference by MSP1(42) on AMA1 antibody responses was also evident when AMA1, MSP1(42) and RTS,S were administered concurrently at separate sites. These results suggest that immune interference may be complex and should be considered for the design of multi-antigen, multi-stage vaccines against malaria.

Objectives: HIV-seropositive pregnant women are more susceptible to malaria than HIV-seronegative women. We assessed whether HIV infection alters maternal and cord plasma malarial antibody responses and the mother-to-infant transfer of malaria antibodies., Methods: We determined plasma levels of maternal and cord antibodies [Immunoglobulin (IgG)] to recombinant malarial proteins [merozoite surface protein 1 (MSP-1(19kD)), the erythrocyte binding antigen (EBA-175)], the synthetic peptides [MSP-2, MSP-3, rhoptry associated protein 1 (RAP-1), and the pre-erythrocytic stage, circumsporozoite protein (NANP)(5)] antigenic determinants of Plasmodium falciparum; and tetanus toxoid (TT) by ELISA among samples of 99 HIV-seropositive mothers, 69 of their infants, 102 HIV-seronegative mothers and 62 of their infants., Results: The prevalence of maternal antibodies to the malarial antigenic determinants ranged from 18% on MSP3 to 91% on EBA-175; in cord plasma it ranged from 13% to 91%, respectively. More than 97% of maternal and cord samples had antibodies to TT. In multivariate analysis, HIV infection was only associated with reduced antibodies to (NANP)(5) in maternal (P=0.001) and cord plasma (P=0.001); and reduced mother-to-infant antibody transfer to (NANP)(5) (P=0.012). This effect of HIV was independent of maternal age, gravidity and placental malaria. No consistent HIV-associated differences were observed for other antigenic determinants., Conclusion: An effect of HIV infection was only observed on one malarial antigenic determinant, suggesting that the increased susceptibility to malaria among HIV-infected pregnant women may not be explained on the basis of their reduced antibody response to malaria antigens.

We compared the needle free jet device device Biojector with syringe/needle as a method to administer a DNA vaccine encoding the Plasmodium falciparum circumsporozoite protein (PfCSP) in albino rabbits. A group of three rabbits was injected by the intramuscular (IM) route using a syringe/needle combination, a second group IM with the Biojector device and a third group both IM and intradermal (ID) using the Biojector. When animals were immunized with the Biojector IM or IM/ID as compared to the syringe/needle IM, we observed 10- and 50-fold greater antibody titers, as measured by enzyme immunoassay (EIA) and indirect fluorescence antibody test (IFAT), respectively. We also observed that the Biojector conferred a greater ability to prime the immune system as compared with the needle. The subsequent boosting of all animals with a recombinant canary pox virus (ALVAC) expressing PfCSP induced significantly higher titers in both Biojector groups of rabbits as compared with the needle and naive animals. These results provided the foundation for a clinical trial using the same regime.

S-Adenosylmethionine (AdoMet) synthetase (SAMS: EC 2.5.1.6) catalyses the formation of AdoMet from methionine and ATP. We have cloned a gene for Plasmodium falciparum AdoMet synthetase (PfSAMS) (GenBank accession no. AF097923), consisting of 1209 base pairs with no introns. The gene encodes a polypeptide (PfSAMS) of 402 amino acids with a molecular mass of 44844 Da, and has an overall base composition of 67% A+T. PfSAMS is probably a single copy gene, and was mapped to chromosome 9. The PfSAMS protein is highly homologous to all other SAMS, including a conserved motif for the phosphate-binding P-loop, HGGGAFSGKD, and the signature hexapeptide, GAGDQG. All the active-site amino acids for the binding of ADP, P(i) and metal ions are similarly preserved, matching entirely those of human hepatic SAMS and Escherichia coli SAMS. Molecular modelling of PfSAMS guided by the X-ray crystal structure of E. coli SAMS indicates that PfSAMS binds ATP/Mg(2+) in a manner similar to that seen in the E. coli SAMS structure. However, the PfSAMS model shows that it can not form tetramers as does E. coli SAMS, and is probably a dimer instead. There was a differential sensitivity towards the inhibition by cycloleucine between the expressed PfSAMS and the human hepatic SAMS with K(i) values of 17 and 10 mM, respectively. Based on phylogenetic analysis using protein parsimony and neighbour-joining algorithms, the malarial PfSAMS is closely related to SAMS of other protozoans and plants.

Candidate malaria vaccines have failed to elicit consistently protective immune responses against challenge with Plasmodium falciparum. NYVAC-Pf7, a highly attenuated vaccinia virus with 7 P. falciparum genes inserted into its genome, was tested in a phase I/IIa safety, immunogenicity, and efficacy vaccine trial in human volunteers. Malaria genes inserted into the NYVAC genome encoded proteins from all stages of the parasite's life cycle. Volunteers received three immunizations of two different dosages of NYVAC-Pf7. The vaccine was safe and well tolerated but variably immunogenic. While antibody responses were generally poor, cellular immune responses were detected in >90% of the volunteers. Of the 35 volunteers challenged with the bite of 5 P. falciparum-infected Anopheles mosquitoes, 1 was completely protected, and there was a significant delay in time to parasite patency in the groups of volunteers who received either the low or high dose of vaccine compared with control volunteers.

Sporozoite surface protein 2 has been identified as a target of malaria vaccines designed to produce protective CD8+ cytotoxic T lymphocytes (CTL) because mice immunized with mastocytoma cells expressing a fragment of Plasmodium yoelii sporozoite surface protein 2 (PySSP2) are protected against malaria by an immune response that requires CD8+ CTL. To define CTL epitopes in the Plasmodium falciparum sporozoite surface protein 2 (PfSSP2), spleen cells (SC) from mice immunized with irradiated sporozoites (irr spz) were stimulated with synthetic peptides, and these effectors were tested for cytolytic activity against peptide-pulsed, major histocompatibility complex (MHC)-matched targets. Two peptides containing CTL epitopes, A6 (Pf SSP2 3D7 214-233) and BH1 (Pf SSP2 3D7 3-11) were identified in bulk cultures of SC from immune C57BL/6 mice, and by production of CTL lines. Immunization with recombinant vaccinia expressing the full length PfSSP2 induced antigen specific, MHC-restricted, CD8+ T cell-dependent cytolytic activity against these two peptides. Finally, CTL were induced by immunization with a bacteria-derived recombinant fragment of PfSSP2 (rPfSSP2) mixed with a liposomal formulation containing a cationic lipid (Lipofectin Reagent, LPF). Induced CTL lysed target cells pulsed with peptide A6 or with LPF/rPfSSP2, but not targets pulsed with only rPfSSP2. These studies demonstrate that CTL specific to PfSSP2 are present in C57BL/6 mice and that immunization with purified rPfSSP2 delivered with LPF induces a cytotoxic T cell response.

Genotypic heterogeneity in the repetitive portion of the circumsporozoite (CS) protein of Plasmodium vivax has been reported from many P. vivax-endemic areas. The objective of this study was to determine if the VK210 and VK247 CS variants of P. vivax differed in their clearance rates following chloroquine (CQ) therapy. One hundred seventy-one cases of P. vivax infection occurring in patients presenting to a research treatment center in Thailand were analyzed. Finger-prick blood samples were collected for microscopy and spotted onto filter paper at presentation and on each of five days of observation through supervised CQ therapy. A portion of the CS gene was amplified from filter paper samples by the polymerase chain reaction (PCR) and genotyped by oligoprobes specific for the VK210 and VK247 CS repeat regions. The mean time to clear parasitemia as determined by thick blood smear was significantly longer for pure VK210 infections (51 hr; 95% confidence interval [CI] 47.4-54, P = 0.006) and mixed infections (53 hr; 95% CI 49.2-56.7, P = 0.0009) as compared with VK247 infections (44 hr; 95% CI 39.8-47.9). Five patients matched for parasitemia, age, sex, and previous malaria experience were selected from each of the three genotype groups in the larger study for further analysis by quantitative PCR of P. vivax genotype-specific DNA during a treatment course. The mean time to clear parasite DNA, as determined by PCR, was significantly slower for VK210 parasites (65 hr; 95% CI 51-79) than for VK247 parasites (47 hr; 95% CI 30-63, P = 0.045).(ABSTRACT TRUNCATED AT 250 WORDS)

EBA-175, erythrocyte binding antigen 175, is a 175-kDa antigen of Plasmodium falciparum which has been shown to be involved in the recognition of erythrocytes by merozoites and may be involved in the process of erythrocyte invasion. Invasion of erythrocytes by Camp strain merozoites is inhibited by pre-treatment of red blood cells by EBA-175 from the heterologous strain, FCR-3. The sequence of the Camp strain has been published and we report here the sequence of the FCR-3 strain. The sequences are nearly identical except for a 423-bp segment in the FCR-3 strain, F-segment, that is not found in the Camp strain and a 342-bp segment, C-segment, present in the Camp strain but not in the FCR-3 strain. The locations of these two segments are different in Camp and FCR-3 EBA-175 genes and there is little DNA or amino acid sequence homology between them. The essentially dimorphic alleles, F-segment and C-segment, are conserved in all isolates examined to date. Evidence of genetic cross-over between the FCR-3 and the Camp EBA-175 genes was not observed in the analysis of a limited number of wild isolates. The continued study of the biological relevance of these sequence divergences in EBA-175 may further elucidate the sequence of events resulting in merozoite invasion of erythrocytes.

The presence in the New World of a variant strain of Plasmodium vivax (VK247) containing a unique circumsporozoite (CS) repeat domain was determined by the detection of antibodies to the variant CS protein and by genetic analysis of the CS gene from field isolates. Whole blood specimens were collected on filter paper from patients infected with P. vivax in Mexico and Peru. Plasmodium vivax DNA was eluted from filter paper samples and the CS gene was amplified by the polymerase chain reaction (PCR) and analyzed for the presence of VK247 or VK210 DNA by oligoprobe hybridization. Sera eluted from a companion filter paper sample were screened for antibodies reactive with the predominant and variant repeat peptides by enzyme-linked immunosorbent assays (ELISA) and with sporozoites by the immunofluorescent antibody (IFA) test. All 24 patients were positive by PCR and oligoprobe hybridization for either VK210 (16 of 24), VK247 (3 of 24), or both (5 of 24). Mixed infections were common (5 of 7) in Peru, but were not observed in the Mexican isolates (0 of 17). All three VK247 infections from Mexico occurred in residents of the foothills above Tapachula (P = 0.02). Of patients with smear-positive P. vivax infection, 42% (10 of 24) had detectable antibodies eluted from dried blood dots that were reactive with the CS protein by IFA or ELISA. These findings establish the widespread distribution of the P. vivax variant CS protein in the New World and indicate that dried blood filter paper samples represent a valuable source of material for the serologic and molecular analysis of plasmodial infections.

We present a rapid and simple system called expression-PCR (E-PCR) for in vitro synthesis of functional protein from genomic or plasmid DNA. A universal promoter was developed containing an untranslated leader sequence from alfalfa mosaic virus directly downstream from the T7 bacteriophage promoter. When this universal promoter is spliced to a DNA segment, it produces a suitable template for in vitro transcription and translation. The DNA to be expressed is first amplified by the PCR using a 5'-primer that incorporates an area homologous to the 3'-end of the universal promoter. The universal promoter and this DNA fragment are mixed and re-amplified in a reaction analogous to splicing by overlap extension, generating a recombinant DNA template that can be transcribed and translated in vitro without further processing. Unlike standard methods for in vitro transcription and translation, E-PCR is not dependent upon specialized transcription vectors, cloning, plasmid isolation and purification, or restriction enzyme sites. This approach has been used to synthesize and examine the biological activity of malaria proteins that are vaccine candidates for Plasmodium falciparum. E-PCR represents a significant improvement over current in vitro expression systems, most notably in its time savings, versatility of gene expression and its compatibility with rapid PCR-based site-directed mutagenesis procedures.

Phenotypic heterogeneity in the repetitive portion of a human malaria circumsporozoite (CS) protein, a major target of candidate vaccines, has been found. Over 14% of clinical cases of uncomplicated Plasmodium vivax malaria at two sites in western Thailand produced sporozoites immunologically distinct from previously characterized examples of the species. Monoclonal antibodies to the CS protein of other P. vivax isolates and to other species of human and simian malarias did not bind to these nonreactive sporozoites, nor did antibodies from monkeys immunized with a candidate vaccine made from the repeat portion of a New World CS protein. The section of the CS protein gene between the conserved regions I and II of a nonreactive isolate contained a nonapeptide repeat, Ala-Asn-Gly-Ala-Gly-Asn-Gln-Pro-Gly, identical at only three amino acid positions with published nonapeptide sequences. This heterogeneity implies that a P. vivax vaccine based on the CS protein repeat of one isolate will not be universally protective.

The content and sequence complexity of the nuclear DNA and messenger RNA for epimastigotes of Trypanosoma cruzi were determined. From analysis of nuclear DNA reassociation studies and microspectrofluorometric measurements of laser induced fluorescence of cellular DNA, T. cruzi is found to be a diploid organism with a nuclear DNA content of 2.5 x 10(8) nucleotide pairs (2.8 x 10(-13) g) and a kinetoplast DNA content of 4.9 x 10(7) nucleotide pairs (5.4 x 10(-14) g). Reassociation kinetics of nuclear DNA of average length 0.4 kb reveals three kinetic components: a moderately repetitive component with a reiteration frequency of 5.1 x 10(3) present in 9% of the fragments, a lowly repetitive component with a reiteration frequency of 32 present in 51% of the fragments, and a single-copy component present in 23% of the fragments. By saturation hybridization of total polysomal RNA to 3H-labeled single-copy DNA, it was determined that 68% of the single-copy DNA was represented in the epimastigote polysomal RNA. This corresponds to ca. 12 000 different mRNA species. Of these, ca. 9000 are present as poly(A)+-RNA, while the remaining 3000 appear not to be polyadenylated. Kinetic analysis of the poly(A)+-RNA population indicates it is composed of at least three classes of RNA's of different abundancy levels: two sequences which occur ca. 3000 per cell, ca. 750 sequences which occur about 20 times per cell, and ca. 15 500 sequences which occur 1-2 times per cell.

Mice immunized intradermally with extracts of Schistosoma mansoni in combination with the adjuvant BCG are significantly protected against subsequent infection with living larval forms of the parasite. Remarkably, these vaccinated animals produce antibodies predominantly against a single parasite protein of molecular weight 97 kilodaltons (Sm-97). A complementary DNA that encodes about half of the Sm-97 molecule has now been cloned and sequenced. Analysis of the deduced amino acid sequence reveals a protein containing periodic repeats of hydrophobic amino acids characteristic of an alpha-helical coiled-coil structure. The deduced amino acid composition of the cloned gene and several properties of the native protein are similar to that of paramyosin, an alpha-helical protein that forms the core for myosin filaments in invertebrate muscle. Paramyosin was isolated from Schistosoma mansoni adult worms and antibodies to Sm-97 were shown to react with this molecule as well as with a known paramyosin from molluscan muscle.

Two clones which contain genes encoding Schistosoma mansoni proteins recognized by immune mouse sera were chosen from cDNA lambda gt11 expression vector library by preselecting clones from the library with rabbit antisera against adult worm phosphate-buffered saline (PBS)-soluble antigens. One clone, MAC 182, codes for part of a Mr 70 000 protein; the other clone, MAC 184, codes for a Mr 27 000 protein. The insert sizes of MAC 182 and MAC 184 are 400 bp and 800 bp, respectively. Both clones express S. mansoni beta-galactosidase fusion proteins as products of the construct. Antibodies from either chronically infected mice or mice vaccinated with irradiated cercariae recognize the MAC 182 fusion protein (MAC 182fp) but not the MAC 184 fusion protein (MAC 184fp). Rabbit antibodies prepared against MAC 182fp immunoprecipitate a Mr 70 000 in vitro translation product from adult mRNA and react in Western blot with a corresponding Mr 70 000 protein present in eggs, cercariae and adult worms but absent in schistosomula. Although the MAC 184fp is not recognized directly by chronic infection or vaccinated mouse antibodies, antisera prepared against the purified fusion protein immunoprecipitate a Mr 27 000 in vitro translation product which also reacts with mouse chronic infection sera. The same Mr 27 000 protein appears to be present in eggs, cercariae, schistosomula and adults as determined by Western blots with rabbit antisera against the MAC 184fp. These results suggest that the S. mansoni polypeptide encoded by the MAC 184 gene, when expressed within a fusion protein, fails to present epitopes normally recognized during natural infection. We propose that these epitopes are conformationally determined and are destroyed when the MAC 184 protein is expressed within beta-galactosidase. This abrogation of conformational epitopes may explain the failure of antibodies from chronically infected or vaccinated mice and rabbits to effectively recognize gene products of certain lambda gt11-fusion protein clones.

The cDNA encoding about half of an antigenic non-surface schistosome parasite protein of Mr 97 K has recently been cloned and sequenced (Lanar, Pearce, James and Sher (1986) Science 234:593-596). Analysis of this sequence, together with the properties of the native protein, reveals that this protein is paramyosin, the hitherto unsequenced core protein of myosin filaments in invertebrate muscle. In this report we analyze in more detail the partial amino acid sequence of schistosome paramyosin and describe electron microscope studies of the native protein and its aggregates. We show a close correspondence between the structures of paramyosin and the myosin rod that is required for these proteins to assemble together in muscle thick filaments.

Using blood from volunteers with sporozoite induced malaria, a comparison was made of the sensitivity and specificity of Giemsa stained thick film examination, in vitro culture, and 4 different DNA probes for detecting parasitemia. Between 9 and 13 days after sporozoite inoculation, patent parasitemia (4-550 parasites/microliters) was detected by thick film examination of 0.5 microliters blood in 7 volunteers. Cultures of 1 ml blood obtained 7 days after sporozoite inoculation were positive in all volunteers who eventually developed patent parasitemia. The DNA hybridization probes detected parasites in only 5-28% of smear- or culture-positive samples.

Trypanosoma cruzi strain Peru was cultivated in the presence of an established cell line of Triatoma infestans embryo cells (TI- 32). Bloodstream trypomastigotes differentiated into amastigote-like cells (first differentiation phase) which multiplied to form large clusters of cells. Because of their clustering nature, a new term, "staphylomastigotes," has been proposed for this stage. After 10 days of cultivation, 90% of the staphylomastigotes underwent differentiation (2nd differentiation phase) to trypomastigotes (approximately 98%) or epimastigotes (approximately 2%). Bloodstream trypomastigotes cultivated without TI-32 cells underwent the first, but not the 2nd differentiation phase, although occasional epimastogotes were seen (less than 1%). The evidence presented suggests that TI-32 cells produce a labile factor(s) important not only for initiation of the 2nd differentiation phase but also for maintaining the parasites in the trypomastigote stage. The pH of the culture medium was not the initiating factor for the 2nd differentiation phase. Infectivity studies indicated that staphylomastigotes were as infective as blood stream trypomastigotes, but that metacyclic trypomastigotes isolated from culture after the 2nd differentiation phase were slightly more infective than bloodstream forms. Electromicrographs of styphylomastigotes do not provide any evidence of exchange of genetic material between cells.

The surface proteins of six stages of Trypanosoma cruzi were labeled by Iodogen-catalized surface iodination and analyzed by one and two dimensional polyacrylamide gel electrophoresis. These stages included bloodstream trypomastigotes, culture-form trypomastigotes, amastigotes, staphylomastigotes, epimastigotes, and metacyclic trypomastigotes. Antigens recognized by serum antibodies were detected by Western blotting against serum from mice hyperimmunized against bloodstream trypomastigotes. Bloodstream trypomastigotes, culture-form trypomastigotes and staphylomastigotes contained several surface proteins of molecular weight (Mr) 90 000 and isoelectric points (pI) between 5.0 and 7.5. Western blotting reveals that at least two proteins of 90 000 Mr represent the major antigens seen on bloodstream trypomastigotes, culture-form trypomastigotes, staphylomastigotes and amastigotes. However, a 90 000 Mr protein was not detected by either Western blotting or surface iodination on epimastigotes or metacyclic trypomastigotes. The major surface proteins on these latter two stages were represented by several 72 000 Mr proteins with pI values between 5.2 and 5.8. An interesting result of this survey is that a 90 000 Mr surface antigen was present on staphylomastigotes, a stage which can be grown in cell free medium.