SPECT ventilation perfusion scanning with the addition of low-dose CT for the investigation of suspected pulmonary embolism.

Single-photon emission computed tomography (SPECT) ventilation perfusion (V/Q) scanning with low-dose computed tomography (LDCT) is an emerging imaging technique for investigation of suspected pulmonary embolism (PE). We aimed to estimate diagnostic utility of the combined technique using results from all patients referred in 2009 compared with final diagnosis and 6-month follow-up status. PE was diagnosed in 28 of 106 patients (26%), including in 2 of 80 (2%) with negative SPECT V/Q and LDCT. The estimated negative predictive value of SPECT V/Q for PE was 97%. LDCT was abnormal in 43 (41%) patients, including 41 patients who had negative SPECT V/Q. In 29 (27%) patients, LDCT provided information on alternative pathologies that accounted for presenting symptoms, and the combined technique had a diagnostic yield of 52%.

The high molecular mass rhoptry protein, RhopH1, is encoded by members of the clag multigene family in Plasmodium falciparum and Plasmodium yoelii.

Malarial merozoite rhoptries contain a high molecular mass protein complex called RhopH. RhopH is composed of three polypeptides, RhopH1, RhopH2, and RhopH3, encoded by distinct genes. Using monoclonal antibody-purified protein complex from both Plasmodium falciparum and Plasmodium yoelii, peptides were obtained by digestion of RhopH1 and their sequence determined either by mass spectrometry or Edman degradation. In both species the genes encoding RhopH1 were identified as members of the cytoadherence linked asexual gene (clag) family. In P. falciparum the family members on chromosome 3 were identified as encoding RhopH1. In P. yoelii two related genes were identified and sequenced. One of the genes, pyrhoph1a, was positively identified as encoding RhopH1 by the peptide analysis and the other gene, pyrhoph1a-p, was at least transcribed. Genes in the clag family present in both parasite species have a number of conserved features. The size and location of the P. yoelii protein complex in the rhoptries was confirmed. The first clag gene identified on chromosome 9 was implicated in cytoadherence, the binding of infected erythrocytes to host endothelial cells; this study shows that other members of the family encode merozoite rhoptry proteins, proteins that may be involved in merozoite-erythrocyte interactions. We propose that the family should be renamed as rhoph1/clag.

The 22 kDa component of the protein complex on the surface of Plasmodium falciparum merozoites is derived from a larger precursor, merozoite surface protein 7.

The gene coding for merozoite surface protein 7 has been identified and sequenced in three lines of Plasmodium falciparum. The gene encodes a 351 amino acid polypeptide that is the precursor of a 22-kDa protein (MSP7(22)) on the merozoite surface and non-covalently associated with merozoite surface protein 1 (MSP1) complex shed from the surface at erythrocyte invasion. A second 19-kDa component of the complex (MSP7(19)) was shown to be derived from MSP7(22) and the complete primary structure of this polypeptide was confirmed by mass spectrometry. The protein sequence contains several predicted helical and two beta elements, but has no similarity with sequences outside the Plasmodium databases. Four sites of sequence variation were identified in MSP7, all within the MSP7(22) region. The MSP7 gene is expressed in mature schizonts, at the same time as other merozoite surface protein genes. It is proposed that MSP7(22) is the result of cleavage by a protease that may also cleave MSP1 and MSP6. A related gene was identified and cloned from the rodent malaria parasite, Plasmodium yoelii YM; at the amino acid level this sequence was 23% identical and 50% similar to that of P. falciparum MSP7.

Inhibitory and blocking monoclonal antibody epitopes on merozoite surface protein 1 of the malaria parasite Plasmodium falciparum.

Merozoite surface protein 1 (MSP-1) is a precursor to major antigens on the surface of Plasmodium spp. merozoites, which are involved in erythrocyte binding and invasion. MSP-1 is initially processed into smaller fragments; and at the time of erythrocyte invasion one of these of 42 kDa (MSP-1(42)) is subjected to a second processing, producing 33 kDa and 19 kDa fragments (MSP-1(33) and MSP-1(19)). Certain MSP-1-specific monoclonal antibodies (mAbs) react with conformational epitopes contained within the two epidermal growth factor domains that comprise MSP-1(19), and are classified as either inhibitory (inhibit processing of MSP-1(42) and erythrocyte invasion), blocking (block the binding and function of the inhibitory mAb), or neutral (neither inhibitory nor blocking). We have mapped the epitopes for inhibitory mAbs 12.8 and 12.10, and blocking mAbs such as 1E1 and 7.5 by using site-directed mutagenesis to change specific amino acid residues in MSP-1(19) and abolish antibody binding, and by using PEPSCAN to measure the reaction of the antibodies with every octapeptide within MSP-1(42). Twenty-six individual amino acid residue changes were made and the effect of each on the binding of mAbs was assessed by Western blotting and BIAcore analysis. Individual changes had either no effect, or reduced, or completely abolished the binding of individual mAbs. No two antibodies had an identical pattern of reactivity with the modified proteins. Using PEPSCAN each mAb reacted with a number of octapeptides, most of which were derived from within the first epidermal growth factor domain, although 1E1 also reacted with peptides spanning the processing site. When the single amino acid changes and the reactive peptides were mapped onto the three-dimensional structure of MSP-1(19), it was apparent that the epitopes for the mAbs could be defined more fully by using a combination of both mutagenesis and PEPSCAN than by either method alone, and differences in the fine specificity of binding for all the different antibodies could be distinguished. The incorporation of several specific amino acid changes enabled the design of proteins that bound inhibitory but not blocking antibodies. These may be suitable for the development of MSP-1-based vaccines against malaria.

Characterization of N-myristoyltransferase from Plasmodium falciparum.

The gene coding for myristoyl-CoA:protein N-myristoyltransferase (NMT) has been cloned from the malaria parasite Plasmodium falciparum. The gene appears to be single copy and mRNA is expressed in asexual blood-stage forms. Comparison of cDNA and genomic sequences identified three small introns. The open reading frame codes for a 410-amino-acid protein and no evidence of forms with an extended N-terminal coding sequence was obtained. Residues important in substrate binding and in the catalytic mechanism in other species are conserved. The protein was expressed from a plasmid in Escherichia coli, partially purified and shown to have enzymic activity using a synthetic peptide substrate. Comparison of the malaria parasite protein with that derived from the human gene showed a different pattern of inhibition by chemical modification. Human NMT activity was inhibited by diethylpyrocarbonate and partially inhibited by iodacetamide, whereas P. falciparum NMT activity was not inhibited by either pre-treatment. Since the enzyme in infectious fungi is a target for potential chemotherapeutic drugs, it should also be investigated in the context of parasitic infections such as that responsible for malaria.

Linkage of exogenous T-cell epitopes to the 19-kilodalton region of Plasmodium yoelii merozoite surface protein 1 (MSP1(19)) can enhance protective immunity against malaria and modulate the immunoglobulin subclass response to MSP1(19).

The degree of protection against Plasmodium yoelii asexual blood stages induced by immunization of mice with the 19-kDa region of merozoite surface protein 1 (MSP1(19)) is H-2 dependent. As a strategy to improve the protection, mouse strains with disparate H-2 haplotypes were immunized with glutathione S-transferase (GST)-MSP1(19) proteins including either a universal T-cell epitope from tetanus toxin (P2) or an I-A(k)-restricted T-cell epitope (P8) from Plasmodium falciparum Pf332. In H-2(k) mice which are poorly protected following immunization with GST-MSP1(19), GST-P2-MSP1(19) significantly improved the protection. In mice partially (H-2(k/b)) or well protected by GST-MSP1(19) (H-2(d) and H-2(b)), P2 did not further increase the protection. However, the protection of H-2(k/b) mice and to some extent H-2(k) mice was improved by immunization with GST-P8-MSP1(19). The magnitudes of immunoglobulin G1 (IgG1) and IgG2a responses in mice immunized with the GST-MSP1(19) variants correlated with low peak parasitemia, indicating a protective capacity of these IgG subclasses. In H-2(k) mice immunized with GST-P2-MSP1(19), both IgG1 and IgG2a responses were significantly enhanced. The epitope P2 appeared to have a general ability to modulate the IgG subclass response since all four mouse strains displayed elevated IgG2a and/or IgG2b levels after immunization with GST-P2-MSP1(19). In contrast, GST-P8-MSP1(19) induced a slight enhancement of IgG responses in H-2(k/b) and H-2(k) mice without any major shift in IgG subclass patterns. The ability to improve the protective immunity elicited by P. yoelii MSP1(19) may have implications for improvement of human vaccines based on P. falciparum MSP1(19).

Antigenic and sequence diversity at the C-terminus of the merozoite surface protein-1 from rodent malaria isolates, and the binding of protective monoclonal antibodies.

Merozoite surface protein-1 (MSP-1) is a major candidate in the development of a vaccine against malaria. Immunisation with a recombinant fusion protein containing the two Plasmodium yoelii MSP-1 C-terminal epidermal growth factor-like domains (MSP-1(19)) can protect mice against homologous but not heterologous challenge, and therefore, antigenic differences resulting from sequence diversity in MSP-1(19) may be crucial in determining the potential of this protein as a vaccine. Representative sequence variants from a number of distinct P. yoelii isolates were expressed in Escherichia coli and the resulting recombinant proteins were screened for binding to a panel of monoclonal antibodies (Mabs) capable of suppressing a P. yoelii YM challenge infection in passive immunisation experiments. The sequence polymorphisms affected the binding of the antibodies to the recombinant proteins. None of the Mabs recognised MSP-1(19) of P. yoelii yoelii 2CL or 33X or P. yoelii nigeriensis N67. The epitopes recognised by the Mabs were further distinguished by their reactivity with the other fusion proteins. The extent of sequence variation in MSP-1(19) among the isolates was extensive, with differences detected at 35 out of the 96 positions compared. Using the 3-dimensional structure of the Plasmodium falciparum MSP-1(19) as a model, the locations of the amino acid substitutions that may affect Mab binding were identified. The DNA sequence of MSP-1(19) from two Plasmodium vinckei isolates was also cloned and the deduced amino acid sequence compared with that in other species.

Merozoite surface protein 1, immune evasion, and vaccines against asexual blood stage malaria.

There is an urgent need for a vaccine against malaria and proteins on the surface of the merozoite are good targets for development as vaccine candidates because they are exposed to antibody. However, it is possible that the parasite has evolved mechanisms to evade a protective immune response to these proteins. Merozoite surface protein 1 (MSP-1) is a candidate for vaccine development and its C-terminal sequence is the target of protective antibody. MSP-1 is cleaved by proteases in two processing steps, the second step releases the bulk of the protein from the surface and goes to completion during successful red blood cell invasion. Antibodies binding to the C-terminus of Plasmodium falciparum MSP-1 can inhibit both the processing and erythrocyte invasion. Other antibodies that bind to either the C-terminal sequence or elsewhere in the molecule are 'blocking' antibodies, which on binding prevent the binding of the inhibitory antibodies. Blocking antibodies are a mechanism of immune evasion, which may be based on antigenic conservation rather than diversity. This mechanism has a number of implications for the study of protective immunity and the development of malaria vaccines, emphasising the need for appropriate functional assays and careful design of the antigen.

Passive immunization with antibodies against three distinct epitopes on Plasmodium yoelii merozoite surface protein 1 suppresses parasitemia.

We have produced monoclonal antibodies against Plasmodium yoelii merozoite surface protein 1 (MSP-1) and have assessed their ability to suppress blood stage parasitemia by passive immunization. Six immunoglobulin G antibodies were characterized in detail: three (B6, D3, and F5) were effective in suppressing a lethal blood stage challenge infection, two (B10 and G3) were partially effective, and one (B4) was ineffective. MSP-1 is the precursor to a complex of polypeptides on the merozoite surface; all of the antibodies bound to this precursor and to an approximately 42-kDa fragment (MSP-142) that is derived from the C terminus of MSP-1. MSP-142 is further cleaved to an N-terminal approximately 33-kDa polypeptide (MSP-133) and a C-terminal approximately 19-kDa polypeptide (MSP-119) comprised of two epidermal growth factor (EGF)-like modules. D3 reacted with MSP-142 but not with either of the constituents MSP-133 and MSP-119, B4 recognized an epitope within the N terminus of MSP-133, and B6, B10, F5, and G3 bound to MSP-119. B10 and G3 bound to epitopes that required both C-terminal EGF-like modules for their formation, whereas B6 and F5 bound to epitopes in the first EGF-like module. These results indicate that at least three distinct epitopes on P. yoelii MSP-1 are recognized by antibodies that suppress parasitemia in vivo.

Definition of T cell epitopes within the 19 kDa carboxylterminal fragment of Plasmodium yoelii merozoite surface protein 1 (MSP1(19)) and their role in immunity to malaria.

MSP1(19) is one of the leading malaria vaccine candidates. However, the mechanism of protection is not clear. To determine whether MSP1(19)-specific effector T cells can control parasitaemia, we analysed the specificity of T cells induced following immunization with recombinant forms of P. yoelii MSP1(19) and asked whether they could protect mice. There was no evidence that effector T cells were capable of protecting since: (1) immunization of mice with yMSP1(19), but not defined epitopes, was able to induce protection; and (2) long term MSP1(19)-specific CD4+ T cell lines were incapable of adoptively transferring protection. In contrast, priming mice with the T cell epitopes resulted in a rapid anamnestic antibody response to MSP1(19) after either challenge with MSP1(19) or parasite. Thus, MSP1(19) contains multiple T cell epitopes but such epitopes are the targets of helper T cells for antibody response but not of identified effector T cells capable of controlling parasitaemia.

Immunization with a recombinant C-terminal fragment of Plasmodium yoelii merozoite surface protein 1 protects mice against homologous but not heterologous P. yoelii sporozoite challenge.

It has been reported previously that immunization with recombinant protein containing the two epidermal growth factor (EGF)-like modules from merozoite surface protein 1 (MSP-1) of Plasmodium yoelii (strain YM) protects mice against a lethal blood-stage challenge with the same parasite strain. Since MSP-1 is expressed in both liver- and blood-stage schizonts and on the surface of merozoites, we evaluated the effectiveness of immunization with recombinant proteins containing either the individual or the two combined EGF-like modules in producing a protective response against a sporozoite challenge. The recombinant protein expressing the combined EGF-like modules of the YM strain protected mice against a homologous sporozoite challenge, and sterile protection, as defined by the absence of detectable blood-stage parasites, was observed in the majority of the mice. In contrast, mice immunized with recombinant P. yoelii YM MSP-1 were not protected against a heterologous challenge with sporozoites from strain 265 BY of P. yoelii. The lack of protection may be explained by differences identified in the amino acid sequences of MSP-1 for the two strains. A recombinant protein containing the two EGF-like modules of MSP-1 from P. yoelii 265 BY was produced and used to immunize mice. These mice were protected against a homologous challenge with sporozoites of P. yoelii 265 BY. The results suggest that a recombinant MSP-1 has potential as a vaccine against malaria, but its efficacy may be limited by sequence polymorphism and selection of variants.

Immunization against the murine malaria parasite Plasmodium yoelii using a recombinant protein with adjuvants developed for clinical use.

Mice vaccinated with a recombinant protein containing the two EGF-like modules of Plasmodium yoelii merozoite surface protein-1 in liposomes or combined with the formulations SBAS2.1 and SBAS2, were protected against a lethal malaria infection. The protection achieved with these adjuvants developed for clinical use was as good as or better than that achieved with Freund's adjuvant. A parasite-specific response was needed for protection. Analysis of the immunoglobulin sub-class response showed that MSP-1-specific IgG1, and to a lesser extent IgG2a and IgG2b, were induced, suggesting that these antibodies were important for protection. Mice passively immunized with serum or purified IgG from vaccinated mice had delayed onset of parasitemia and were able to control the infection.

Cytokines and antibody subclass associated with protective immunity against blood-stage malaria in mice vaccinated with the C terminus of merozoite surface protein 1 plus a novel adjuvant.

A blood-stage malaria antigen comprising the C terminus of merozoite surface protein 1 fused to glutathione S-transferase, combined with an adjuvant formulation containing squalane, Tween 80, and pluronic L121 (AF), administered subcutaneously protected mice against death from a lethal Plasmodium yoelii infection. The protection induced by this antigen-adjuvant combination was compared with that induced by the antigen plus saponin in terms of survival from the lethal infection and clearance of parasitemia. The levels of gamma interferon and interleukin-4 in spleens were measured as indicators of Th1 and Th2 cell activation, and antibody classes and subclasses were determined by immunofluorescence. With a 10-micrograms dose of antigen and AF as adjuvant, all mice recovered, but with saponin as the adjuvant, there were only a few survivors. With 30 micrograms of antigen plus AF, the peak parasitemias were 10-fold lower than those with 10 micrograms; with saponin, survival was slightly improved. The levels of both gamma interferon and interleukin-4 rose more rapidly and to higher levels with AF as the adjuvant than with saponin, and the same was true for immunoglobulin G1 (IgG1), IgG2a, and IgG2b subclasses. Thus, in terms of both cytokine production and antibody levels, AF is a more potent adjuvant for a malaria vaccine than is saponin.

The combined epidermal growth factor-like modules of Plasmodium yoelii Merozoite Surface Protein-1 are required for a protective immune response to the parasite.

We have reported previously that immunization with a bacterial recombinant protein containing the two epidermal growth factor (EGF)-like modules of Plasmodium yoelii Merozoite Surface Protein-1 (MSP-1) protected mice against challenge with this malaria parasite. Bacterial plasmids containing sequences coding for the individual modules fused to glutathione S-transferase (GST) have now been made. The fusion protein containing the combined EGF-like modules was recognized by anti-parasite antibodies and was immunogenic, producing high titre anti-parasite and anti-GST antibodies. In contrast, fusion proteins containing the two individual EGF-like modules reacted poorly with the natural antibodies and their proteins, as well as a simple mixture of them, induced low levels of anti-parasite antibodies despite producing high levels of anti-GST antibody. Antibodies raised to the recombinant proteins recognized the 230 kDa MSP-1. Groups of mice immunized with the different recombinant proteins were challenged with parasites: protection was observed in the group which had received the recombinant protein containing both modules but not in those groups immunized with the individual modules, either alone or as a mixture. These results suggest that there are important structural determinants formed by the two modules together, which are not present in either of the individual domains alone, and which are responsible for the immunogenicity of the protein or are the target of protective antibodies.

Immunization against malaria with a recombinant protein.

We have expressed in bacteria the C-terminal part of Plasmodium yoelii merozoite surface protein-1 (MSP1) containing the two epidermal growth factor-like domains. The protein, either alone or fused to glutathione S-transferase, was highly effective as a vaccine and protected mice against challenge infection. Reduction and alkylation abolished the protection obtained with the protein. This shows for the first time the absolute requirement of the disulphide-bonded conformation for immunogenicity. In a short term experiment, mice were protected against a massive challenge. The immunity was effective at the time of merozoite release/reinvasion. Recombinant protein based on this part of MSP1 may be suitable as a vaccine against malaria.

A malaria merozoite surface protein (MSP1)-structure, processing and function.

Merozoite surface protein-1 (MSP-1, also referred to as P195, PMMSA or MSA 1) is one of the most studied of all malaria proteins. The protein is found in all malaria species investigated and structural studies on the gene indicate that parts of the molecule are well-conserved. Studies on Plasmodium falciparum have shown that the protein is in a processed form on the merozoite surface, a result of proteolytic cleavage of the large precursor molecule. Recent studies have identified some of these cleavage sites. During invasion of the new red cell most of the MSP1 molecule is shed from the parasite surface except for a small C-terminal fragment which can be detected in ring stages. Analysis of the structure of this fragment suggests that it contains two growth factor-like domains that may have a functional role.

Proteolytic processing of the Plasmodium falciparum merozoite surface protein-1 produces a membrane-bound fragment containing two epidermal growth factor-like domains.

The amino-terminal sequence has been obtained for 2 fragments of the Plasmodium falciparum T9/94 merozoite surface protein precursor (PfMSP1) and these have been compared with the sequence predicted from the gene. These data define the position of these fragments in the precursor and indicate that the C-terminal sequence which is carried into the red cell during invasion consists of 2 epidermal growth factor (EGF)-like domains. A homologous cleavage sequence and domain structure can be identified in the MSP1 molecules of other malarial species. In addition the results suggest that the smaller fragment is not N-glycosylated.

Mitochondrial DNA of the human malarial parasite Plasmodium falciparum.

Covalently closed circular DNA molecules were isolated from Plasmodium falciparum total DNA by isopycnic centrifugation in CsCl gradients containing either ethidium bromide or 2',6-diamidino-2-phenylindole. The circular molecules had an average contour length of 11.1 +/- 0.5 micron, similar to the analogous molecules previously isolated from the simian malaria parasite P. knowlesi. Both circular molecules shared considerable sequence homology and conserved restriction sites. The nucleotide sequence of one 936 bp fragment of the P. falciparum molecule was determined and identified, by a data base homology search, as part of a mitochondrial small rRNA subunit, thus confirming the mitochondrial origin of the circular DNAs of both malarial species.