Proteomic and genetic analyses demonstrate that Plasmodium berghei blood stages export a large and diverse repertoire of proteins.

Malaria parasites actively remodel the infected red blood cell (irbc) by exporting proteins into the host cell cytoplasm. The human parasite Plasmodium falciparum exports particularly large numbers of proteins, including proteins that establish a vesicular network allowing the trafficking of proteins onto the surface of irbcs that are responsible for tissue sequestration. Like P. falciparum, the rodent parasite P. berghei ANKA sequesters via irbc interactions with the host receptor CD36. We have applied proteomic, genomic, and reverse-genetic approaches to identify P. berghei proteins potentially involved in the transport of proteins to the irbc surface. A comparative proteomics analysis of P. berghei non-sequestering and sequestering parasites was used to determine changes in the irbc membrane associated with sequestration. Subsequent tagging experiments identified 13 proteins (Plasmodium export element (PEXEL)-positive as well as PEXEL-negative) that are exported into the irbc cytoplasm and have distinct localization patterns: a dispersed and/or patchy distribution, a punctate vesicle-like pattern in the cytoplasm, or a distinct location at the irbc membrane. Members of the PEXEL-negative BIR and PEXEL-positive Pb-fam-3 show a dispersed localization in the irbc cytoplasm, but not at the irbc surface. Two of the identified exported proteins are transported to the irbc membrane and were named erythrocyte membrane associated proteins. EMAP1 is a member of the PEXEL-negative Pb-fam-1 family, and EMAP2 is a PEXEL-positive protein encoded by a single copy gene; neither protein plays a direct role in sequestration. Our observations clearly indicate that P. berghei traffics a diverse range of proteins to different cellular locations via mechanisms that are analogous to those employed by P. falciparum. This information can be exploited to generate transgenic humanized rodent P. berghei parasites expressing chimeric P. berghei/P. falciparum proteins on the surface of rodent irbc, thereby opening new avenues for in vivo screening adjunct therapies that block sequestration.

KAI407, a potent non-8-aminoquinoline compound that kills Plasmodium cynomolgi early dormant liver stage parasites in vitro.

Preventing relapses of Plasmodium vivax malaria through a radical cure depends on use of the 8-aminoquinoline primaquine, which is associated with safety and compliance issues. For future malaria eradication strategies, new, safer radical curative compounds that efficiently kill dormant liver stages (hypnozoites) will be essential. A new compound with potential radical cure activity was identified using a low-throughput assay of in vitro-cultured hypnozoite forms of Plasmodium cynomolgi (an excellent and accessible model for Plasmodium vivax). In this assay, primary rhesus hepatocytes are infected with P. cynomolgi sporozoites, and exoerythrocytic development is monitored in the presence of compounds. Liver stage cultures are fixed after 6 days and stained with anti-Hsp70 antibodies, and the relative proportions of small (hypnozoite) and large (schizont) forms relative to the untreated controls are determined. This assay was used to screen a series of 18 known antimalarials and 14 new non-8-aminoquinolines (preselected for blood and/or liver stage activity) in three-point 10-fold dilutions (0.1, 1, and 10 µM final concentrations). A novel compound, designated KAI407 showed an activity profile similar to that of primaquine (PQ), efficiently killing the earliest stages of the parasites that become either primary hepatic schizonts or hypnozoites (50% inhibitory concentration [IC50] for hypnozoites, KAI407, 0.69 µM, and PQ, 0.84 µM; for developing liver stages, KAI407, 0.64 µM, and PQ, 0.37 µM). When given as causal prophylaxis, a single oral dose of 100 mg/kg of body weight prevented blood stage parasitemia in mice. From these results, we conclude that KAI407 may represent a new compound class for P. vivax malaria prophylaxis and potentially a radical cure.

Diversity covering AMA1-MSP119 fusion proteins as malaria vaccines.

To overcome polymorphism in the malaria vaccine candidate Plasmodium falciparum apical membrane antigen 1 (PfAMA1), fusion protein chimeras comprised of three diversity-covering (DiCo) PfAMA1 molecules (D1, D2, and D3) and two allelic variants of the C-terminal 19-kDa region of merozoite surface protein 1 (MSP119) (variants M1 and M2) were generated. A mixture of fusion proteins (D1M1/D2M2D3) and the D1M1D2M2D3 fusion were compared to a single-unit mixture (D1/D2/D3/M1) in an immunological study in groups of rabbits. Following immunization, titers of antibodies (Abs) against four naturally occurring PfAMA1 alleles were high for all groups, as were growth inhibition assay (GIA) levels against two antigenically distinct laboratory parasite strains. Fusion of AMA1 to MSP119 did not suppress levels of antibodies against the AMA1 component. In addition, the breadth of antibody responses was unaffected. Anti-AMA1 antibodies were largely responsible for parasite growth inhibition, as shown in reversal-of-inhibition experiments by adding competing AMA1 antigen. For all groups, titration of the MSP119 antigen into the GIA led to only a small decrease in parasite inhibition, although titers of antibodies against MSP119 were increased 15-fold for the groups immunized with fusion proteins. GIA with affinity-purified anti-MSP119 antibodies showed that the 50% inhibitory concentrations of the anti-MSP119 antibody preparations were in the same order of magnitude for all animals tested, leading to the conclusion that fusing MSP119 to PfAMA1 leads to a small but significant increase in functional antibody levels. This study shows that combination of multiple vaccine candidates in fusion proteins may lead to improved characteristics of the vaccine.

The RON2-AMA1 interaction is a critical step in moving junction-dependent invasion by apicomplexan parasites.

Obligate intracellular Apicomplexa parasites share a unique invasion mechanism involving a tight interaction between the host cell and the parasite surfaces called the moving junction (MJ). The MJ, which is the anchoring structure for the invasion process, is formed by secretion of a macromolecular complex (RON2/4/5/8), derived from secretory organelles called rhoptries, into the host cell membrane. AMA1, a protein secreted from micronemes and associated with the parasite surface during invasion, has been shown in vitro to bind the MJ complex through a direct association with RON2. Here we show that RON2 is inserted as an integral membrane protein in the host cell and, using several interaction assays with native or recombinant proteins, we define the region that binds AMA1. Our studies were performed both in Toxoplasma gondii and Plasmodium falciparum and although AMA1 and RON2 proteins have diverged between Apicomplexa species, we show an intra-species conservation of their interaction. More importantly, invasion inhibition assays using recombinant proteins demonstrate that the RON2-AMA1 interaction is crucial for both T. gondii and P. falciparum entry into their host cells. This work provides the first evidence that AMA1 uses the rhoptry neck protein RON2 as a receptor to promote invasion by Apicomplexa parasites.

Plasmodium falciparum 19-kilodalton merozoite surface protein 1 (MSP1)-specific antibodies that interfere with parasite growth in vitro can inhibit MSP1 processing, merozoite invasion, and intracellular parasite development.

Merozoite surface protein 1 (MSP1) is a target for malaria vaccine development. Antibodies to the 19-kDa carboxy-terminal region referred to as MSP1(19) inhibit erythrocyte invasion and parasite growth, with some MSP1-specific antibodies shown to inhibit the proteolytic processing of MSP1 that occurs at invasion. We investigated a series of antibodies purified from rabbits immunized with MSP1(19) and AMA1 recombinant proteins for their ability to inhibit parasite growth, initially looking at MSP1 processing. Although significant inhibition of processing was mediated by several of the antibody samples, there was no clear relationship with overall growth inhibition by the same antibodies. However, no antibody samples inhibited processing but not invasion, suggesting that inhibition of MSP1 processing contributes to but is not the only mechanism of antibody-mediated inhibition of invasion and growth. Examining other mechanisms by which MSP1-specific antibodies inhibit parasite growth, we show that MSP1(19)-specific antibodies are taken up into invaded erythrocytes, where they persist for significant periods and result in delayed intracellular parasite development. This delay may result from antibody interference with coalescence of MSP1(19)-containing vesicles with the food vacuole. Antibodies raised against a modified recombinant MSP1(19) sequence were more efficient at delaying intracellular growth than those to the wild-type protein. We propose that antibodies specific for MSP1(19) can mediate inhibition of parasite growth by at least three mechanisms: inhibition of MSP1 processing, direct inhibition of invasion, and inhibition of parasite development following invasion. The balance between mechanisms may be modulated by modifying the immunogen used to induce the antibodies.

Safety and immunogenicity of multi-antigen AMA1-based vaccines formulated with CoVaccine HT™ and Montanide ISA 51 in rhesus macaques.

BACKGROUND: Increasing the breadth of the functional antibody response through immunization with Plasmodium falciparum apical membrane antigen 1 (PfAMA1) multi-allele vaccine formulations has been demonstrated in several rodent and rabbit studies. This study assesses the safety and immunogenicity of three PfAMA1 Diversity-Covering (DiCo) vaccine candidates formulated as an equimolar mixture (DiCo mix) in CoVaccine HT™ or Montanide ISA 51, as well as that of a PfAMA1-MSP119 fusion protein formulated in Montanide ISA 51. METHODS: Vaccine safety in rhesus macaques was monitored by animal behaviour observation and assessment of organ and systemic functions through clinical chemistry and haematology measurements. The immunogenicity of vaccine formulations was assessed by enzyme-linked immunosorbent assays and in vitro parasite growth inhibition assays with three culture-adapted P. falciparum strains. RESULTS: These data show that both adjuvants were well tolerated with only transient changes in a few of the chemical and haematological parameters measured. DiCo mix formulated in CoVaccine HT™ proved immunologically and functionally superior to the same candidate formulated in Montanide ISA 51. Immunological data from the fusion protein candidate was however difficult to interpret as four out of six immunized animals were non-responsive for unknown reasons. CONCLUSIONS: The study highlights the safety and immunological benefits of DiCo mix as a potential human vaccine against blood stage malaria, especially when formulated in CoVaccine HT™, and adds to the accumulating data on the specificity broadening effects of DiCo mix.

Immunization with different PfAMA1 alleles in sequence induces clonal imprint humoral responses that are similar to responses induced by the same alleles as a vaccine cocktail in rabbits.

BACKGROUND: Antibodies to key Plasmodium falciparum surface antigens have been shown to be important effectors that mediate clinical immunity to malaria. The cross-strain fraction of anti-malarial antibodies may however be required to achieve strain-transcending immunity. Such antibody responses against Plasmodium falciparum apical membrane antigen 1 (PfAMA1), a vaccine target molecule that is expressed in both liver and blood stages of the parasite, can be elicited through immunization with a mixture of allelic variants of the parasite molecule. Cross-strain antibodies are most likely elicited against epitopes that are shared by the allelic antigens in the vaccine cocktail. METHODS: A standard competition ELISA was used to address whether the antibody response can be further focused on shared epitopes by exclusively boosting these common determinants through immunization of rabbits with different PfAMA1 alleles in sequence. The in vitro parasite growth inhibition assay was used to further evaluate the functional effects of the broadened antibody response that is characteristic of multi-allele vaccine strategies. RESULTS: A mixed antigen immunization protocol elicited humoral responses that were functionally similar to those elicited by a sequential immunization protocol (p > 0.05). Sequential exposure to the different PfAMA1 allelic variants induced immunological recall of responses to previous alleles and yielded functional cross-strain antibodies that would be capable of optimal growth inhibition of variant parasites at high enough concentrations. CONCLUSIONS: These findings may have implications for the current understanding of the natural acquisition of clinical immunity to malaria as well as for rational vaccine design.

Malaria infection by sporozoite challenge induces high functional antibody titres against blood stage antigens after a DNA prime, poxvirus boost vaccination strategy in Rhesus macaques.

BACKGROUND: A DNA prime, poxvirus (COPAK) boost vaccination regime with four antigens, i.e. a combination of two Plasmodium knowlesi sporozoite (csp/ssp2) and two blood stage (ama1/msp142) genes, leads to self-limited parasitaemia in 60% of rhesus monkeys and survival from an otherwise lethal infection with P. knowlesi. In the present study, the role of the blood stage antigens in protection was studied in depth, focusing on antibody formation against the blood stage antigens and the functionality thereof. METHODS: Rhesus macaques were immunized with the four-component vaccine and subsequently challenged i.v. with 100 P. knowlesi sporozoites. During immunization and challenge, antibody titres against the two blood stage antigens were determined, as well as the in vitro growth inhibition capacity of those antibodies. Antigen reversal experiments were performed to determine the relative contribution of antibodies against each of the two blood stage antigens to the inhibition. RESULTS: After vaccination, PkAMA1 and PkMSP119 antibody titres in vaccinated animals were low, which was reflected in low levels of inhibition by these antibodies as determined by in vitro inhibition assays. Interestingly, after sporozoite challenge antibody titres against blood stage antigens were boosted over 30-fold in both protected and not protected animals. The in vitro inhibition levels increased to high levels (median inhibitions of 59% and 56% at 6 mg/mL total IgG, respectively). As growth inhibition levels were not significantly different between protected and not protected animals, the ability to control infection appeared cannot be explained by GIA levels. Judged by in vitro antigen reversal growth inhibition assays, over 85% of the inhibitory activity of these antibodies was directed against PkAMA1. CONCLUSIONS: This is the first report that demonstrates that a DNA prime/poxvirus boost vaccination regimen induces low levels of malaria parasite growth inhibitory antibodies, which are boosted to high levels upon challenge. No association could, however, be established between the levels of inhibitory capacity in vitro and protection, either after vaccination or after challenge.

Suppression of Plasmodium cynomolgi in rhesus macaques by coinfection with Babesia microti.

Both Plasmodium and Babesia species are intraerythrocytic protozoans that infect a wide range of hosts, including humans, and they elicit similar inflammatory responses and clinical manifestations that differ markedly in severity. We recently reported that a rhesus macaque that was chronically infected with Babesia microti was able to control infection with Plasmodium cynomolgi (a parasite of macaques with characteristics very similar to those of Plasmodium vivax) better than naïve monkeys. To confirm this and to investigate the underlying immunopathology, six naïve rhesus monkeys were infected with B. microti. After 24 days, four of these monkeys and four naïve rhesus monkeys were challenged with P. cynomolgi blood-stage parasites. B. microti persisted at low levels in all monkeys, and the clinical parameters were comparable to those of noninfected controls. There was a significant decrease in P. cynomolgi parasitemia in animals coinfected with B. microti compared to the parasitemia in animals infected with P. cynomolgi alone. This decrease in P. cynomolgi parasitemia correlated with increases in the levels of proinflammatory monocytes at the time of P. cynomolgi infection and with higher C-reactive protein (CRP) serum levels 1 week after malaria infection. Therefore, we conclude that ongoing infection with B. microti parasites leads to suppression of malaria infection.

Deep coverage mouse red blood cell proteome: a first comparison with the human red blood cell.

Mice have close genetic/physiological relationships to humans, breed rapidly, and can be genetically modified, making them the most used mammal in biomedical research. Because the red blood cell (RBC) is the sole gas transporter in vertebrates, diseases of the RBC are frequently severe; much research has therefore focused on RBC and cardiovascular disorders of mouse and humans. RBCs also host malaria parasites. Recently we presented an in-depth proteome for the human RBC. Here we present directly comparable data for the mouse RBC as membrane-only, soluble-only, and combined membrane-bound/soluble proteomes (comprising, respectively, 247, 232, and 165 proteins). All proteins were identified, validated, and categorized in terms of subcellular localization, protein family, and function, and in comparison with the human RBC, were classified as orthologs, family-related, or unique. Splice isoforms were identified, and polypeptides migrating with anomalous apparent molecular weights were grouped into putatively ubiquitinated or partially degraded complexes. Overall there was close concordance between mouse and human proteomes, confirming the unexpected RBC complexity. Several novel findings in the human proteome have been confirmed here. This comparison sheds light on several open issues in RBC biology and provides a departure point for more comprehensive understanding of RBC function.

Statistical model to evaluate in vivo activities of antimalarial drugs in a Plasmodium cynomolgi-macaque model for Plasmodium vivax malaria.

Preclinical animal models informing antimalarial drug development are scarce. We have used asexual erythrocytic Plasmodium cynomolgi infections of rhesus macaques to model Plasmodium vivax during preclinical development of compounds targeting parasite phospholipid synthesis. Using this malaria model, we accumulated data confirming highly reproducible infection patterns, with self-curing parasite peaks reproducibly preceding recrudescence peaks. We applied nonlinear mixed-effect (NLME) models, estimating treatment effects in three drug studies: G25 (injected) and the bisthiazolium prodrugs TE4gt and TE3 (oral). All compounds fully cured P. cynomolgi-infected macaques, with significant effects on parasitemia height and time of peak. Although all three TE3 doses tested were fully curative, NLME models discriminated dose-dependent differential pharmacological antimalarial activity. By applying NLME modeling treatment effects are readily quantified. Such drug development studies are more informative and contribute to reduction and refinement in animal experimentation.

A diversity-covering approach to immunization with Plasmodium falciparum apical membrane antigen 1 induces broader allelic recognition and growth inhibition responses in rabbits.

Plasmodium falciparum apical membrane antigen 1 (PfAMA1), a candidate malaria vaccine, is polymorphic. This polymorphism is believed to be generated predominantly under immune selection pressure and, as a result, may compromise attempts at vaccination. Alignment of 355 PfAMA1 sequences shows that around 10% of the 622 amino acid residues can vary between alleles and that linkages between polymorphic residues occur. Using this analysis, we have designed three diversity-covering (DiCo) PfAMA1 sequences that take account of these linkages and, when taken together, on average incorporate 97% of amino acid variability observed. For each of the three DiCo sequences, a synthetic gene was constructed and used to transform the methylotrophic yeast Pichia pastoris, allowing recombinant expression. All three DiCo proteins were reactive with the reduction-sensitive monoclonal antibody 4G2, suggesting the DiCo sequences had conformations similar to those of naturally occurring PfAMA1. Rabbits were immunized with FVO strain PfAMA1 or with the DiCo proteins either individually or as a mixture. Antibody titers and the ability to inhibit parasite growth in vitro were determined. Animals immunized with the DiCo mix performed similarly to animals immunized with FVO AMA1 when measured against FCR3 strain parasites but outperformed animals immunized with FVO AMA1 when assessed against other strains. The levels of growth inhibition (approximately 70%) induced by the mix of three DiCo proteins were comparable for FVO, 3D7, and HB3, suggesting that a considerable degree of diversity in AMA1 is adequately covered. This suggests that vaccines based upon the DiCo mix approach provide a broader functional immunity than immunization with a single allele.

Breadth and magnitude of antibody responses to multiple Plasmodium falciparum merozoite antigens are associated with protection from clinical malaria.

Individuals living in areas where malaria is endemic are repeatedly exposed to many different malaria parasite antigens. Studies on naturally acquired antibody-mediated immunity to clinical malaria have largely focused on the presence of responses to individual antigens and their associations with decreased morbidity. We hypothesized that the breadth (number of important targets to which antibodies were made) and magnitude (antibody level measured in a random serum sample) of the antibody response were important predictors of protection from clinical malaria. We analyzed naturally acquired antibodies to five leading Plasmodium falciparum merozoite-stage vaccine candidate antigens, and schizont extract, in Kenyan children monitored for uncomplicated malaria for 6 months (n = 119). Serum antibody levels to apical membrane antigen 1 (AMA1) and merozoite surface protein antigens (MSP-1 block 2, MSP-2, and MSP-3) were inversely related to the probability of developing malaria, but levels to MSP-1(19) and erythrocyte binding antigen (EBA-175) were not. The risk of malaria was also inversely associated with increasing breadth of antibody specificities, with none of the children who simultaneously had high antibody levels to five or more antigens experiencing a clinical episode (17/119; 15%; P = 0.0006). Particular combinations of antibodies (AMA1, MSP-2, and MSP-3) were more strongly predictive of protection than others. The results were validated in a larger, separate case-control study whose end point was malaria severe enough to warrant hospital admission (n = 387). These findings suggest that under natural exposure, immunity to malaria may result from high titers antibodies to multiple antigenic targets and support the idea of testing combination blood-stage vaccines optimized to induce similar antibody profiles.

Apical membrane antigen 1: a malaria vaccine candidate in review.

Apical membrane antigen 1 (AMA1) is a micronemal protein of apicomplexan parasites that appears to be essential during the invasion of host cells. Immune responses to Plasmodium AMA1 can have profound parasite-inhibitory effects, both as measured in vitro and in animal challenge models, suggesting AMA1 as a potential vaccine component. However, AMA1 is polymorphic, probably as a result of immune selection operating on an important target of naturally occurring immunity. The current understanding of AMA1 will be presented, particularly in relation to the vaccine potential of AMA1 and the approaches being taken towards clinical development.

Cross-reactivity studies of an anti-Plasmodium vivax apical membrane antigen 1 monoclonal antibody: binding and structural characterisation.

Apical membrane antigen 1 (AMA1) has an important, but as yet uncharacterised, role in host cell invasion by the malaria parasite, Plasmodium. The protein, which is quite conserved between Plasmodium species, comprises an ectoplasmic region, a single transmembrane segment and a small cytoplasmic domain. The ectoplasmic region, which can induce protective immunity in animal models of human malaria, is a leading vaccine candidate that has entered clinical trials. The monoclonal antibody F8.12.19, raised against the recombinant ectoplasmic region of AMA1 from Plasmodium vivax, cross-reacts with homologues from Plasmodium knowlesi, Plasmodium cynomolgi, Plasmodium berghei and Plasmodium falciparum, as shown by immunofluorescence assays on mature schizonts. The binding of F8.12.19 to recombinant AMA1 from both P. vivax and P. falciparum was measured by surface plasmon resonance, revealing an apparent affinity constant that is about 100-fold weaker for the cross-reacting antigen when compared to the cognate antigen. Crystal structure analysis of Fab F8.12.19 complexed to AMA1 from P. vivax and P. falciparum shows that the monoclonal antibody recognises a discontinuous epitope located on domain III of the ectoplasmic region, the major component being a loop containing a cystine knot. The structures provide a basis for understanding the cross-reactivity. Antibody contacts are made mainly to main-chain and invariant side-chain atoms of AMA1; contact antigen residues that differ in sequence are located at the periphery of the antigen-binding site and can be accommodated at the interface between the two components of the complex. The implications for AMA1 vaccine development are discussed.

Malaria vaccine-related benefits of a single protein comprising Plasmodium falciparum apical membrane antigen 1 domains I and II fused to a modified form of the 19-kilodalton C-terminal fragment of merozoite surface protein 1.

We show that the smallest module of Plasmodium falciparum AMA1 (PfAMA1) that can be expressed in the yeast Pichia pastoris while retaining the capacity to induce high levels of parasite-inhibitory antibodies comprises domains I and II. Based on this, two fusion proteins, differing in the order of the modules, were developed. Each comprised one module of PfAMA1 (FVO strain, amino acids [aa] 97 to 442) (module A) and one module of PfMSP1(19) (Wellcome strain, aa 1526 to 1621) (module Mm) in which a cystine had been removed to improve immune responses. Both fusion proteins retained the antigenicity of each component and yielded over 30 mg/liter purified protein under fed-batch fermentation. Rabbits immunized with purified fusion proteins MmA and AMm had up to eightfold-higher immune responses to MSP1(19) than those of rabbits immunized with module Mm alone or Mm mixed with module A. In terms of parasite growth inhibition, fusion did not diminish the induction of inhibitory antibodies compared with immunization with module A alone or module A mixed with module Mm, and fusion outperformed antibodies induced by immunization with module M or Mm alone. When tested against parasites expressing AMA1 heterologous to the immunogen, antibodies to the fusion proteins inhibited parasite growth to a greater extent than did antibodies either to the individual antigens or to the mixture. These results suggest that compared with the individual modules delivered separately or as a mixture, fusion proteins containing these two modules offer the potential for significant vaccine-related advantages in terms of ease of production, immunogenicity, and functionality.

Differential evidence of natural selection on two leading sporozoite stage malaria vaccine candidate antigens.

Experimental malaria vaccines based on two sporozoite stage candidate antigens of Plasmodium falciparum, the circumsporozoite protein (CSP) and thrombospondin-related adhesive protein (TRAP), have undergone clinical trials of efficacy. The relevance of naturally existing polymorphism in these molecules remains unknown. Sequence polymorphism in the genes encoding these antigens was studied in a Gambian population (sample of 48 trap and 44 csp gene sequences) to test for signatures of selection that would result from naturally acquired immunity. Allele frequency distributions were analyzed and compared with data from another population (in Thailand). Patterns of non-synonymous and synonymous polymorphism in P. falciparum and in Plasmodium vivax were compared with divergence from related species. Results indicate that polymorphism in TRAP is under strong selection for amino acid sequence diversity and that allele frequencies are under balancing selection within the Gambian P. falciparum population. There was no such evidence for CSP, calling into question the idea that most polymorphisms in this gene are under immune selection. There was a weak trend for regions known to encode T cell epitopes to have slightly higher indices suggesting balancing selection. Overall, the results predict more allele-specific immunity to TRAP than to CSP and should be considered in design and efficacy testing of vaccine candidates based on these antigens.

Variable BCG efficacy in rhesus populations: Pulmonary BCG provides protection where standard intra-dermal vaccination fails.

M.bovis BCG vaccination against tuberculosis (TB) notoriously displays variable protective efficacy in different human populations. In non-human primate studies using rhesus macaques, despite efforts to standardise the model, we have also observed variable efficacy of BCG upon subsequent experimental M. tuberculosis challenge. In the present head-to-head study, we establish that the protective efficacy of standard parenteral BCG immunisation varies among different rhesus cohorts. This provides different dynamic ranges for evaluation of investigational vaccines, opportunities for identifying possible correlates of protective immunity and for determining why parenteral BCG immunisation sometimes fails. We also show that pulmonary mucosal BCG vaccination confers reduced local pathology and improves haematological and immunological parameters post-infection in animals that are not responsive to induction of protection by standard intra-dermal BCG. These results have important implications for pulmonary TB vaccination strategies in the future.

Production, Quality Control, Stability and Pharmacotoxicity of a Malaria Vaccine Comprising Three Highly Similar PfAMA1 Protein Molecules to Overcome Antigenic Variation.

Plasmodium falciparum apical membrane antigen 1 (PfAMA1) is a leading asexual blood stage vaccine candidate for malaria. In preparation for clinical trials, three Diversity Covering (DiCo) PfAMA1 ectodomain proteins, designed to overcome the intrinsic polymorphism that is present in PfAMA1, were produced under Good Manufacturing Practice (GMP) in Pichia pastoris. Using identical methodology, the 3 strains were cultivated in 70-L scale fed-batch fermentations and PfAMA1-DiCos were purified by two chromatography steps, an ultrafiltration/diafiltration procedure and size exclusion chromatography, resulting in highly pure (>95%) PfAMA1-DiCo1, PfAMA1 DiCo2 and PfAMA1 DiCo3, with final yields of 1.8, 1.9 and 1.3 gram, respectively. N-terminal determinations showed that approximately 50% of each of the proteins lost 12 residues from their N-terminus, in accordance with SDS-PAGE (2 main bands) and MS-data. Under reducing conditions a site of limited proteolytic cleavage within a disulphide bonded region became evident. The three proteins quantitatively bound to the mAb 4G2 that recognizes a conformational epitope, suggesting proper folding of the proteins. The lyophilized Drug Product (1:1:1 mixture of PfAMA1-DiCo1, DiCo2, DiCo3) fulfilled all pre-set release criteria (appearance, dissolution rate, identity, purity, protein content, moisture content, sub-visible particles, immuno-potency (after reconstitution with adjuvant), abnormal toxicity, sterility and endotoxin), was stable in accelerated and real-time stability studies at -20°C for over 24 months. When formulated with adjuvants selected for clinical phase I evaluation, the Drug Product did not show adverse effect in a repeated-dose toxicity study in rabbits. The Drug Product has entered a phase Ia/Ib clinical trial.

Orthologous gene sequences of merozoite surface protein 1 (MSP1) from Plasmodium reichenowi and P. gallinaceum confirm an ancient divergence of P. falciparum alleles.

Merozoite surface protein 1 (MSP 1) of Plasmodium falciparum has a major allelic dimorphism in the majority of its sequence, the origin and significance of which is obscure. Here, the cloning and sequencing of the msp1 gene from P. reichenowi (a chimpanzee parasite that is the nearest relative of P. falciparum) and P. gallinaceum (a malaria parasite of birds) is reported. P. reichenowi msp1 is most closely related to one allelic type (K1) of P. falciparum. The other P. falciparum major allelic type (MAD20) is very divergent from these sequences, although not as divergent as msp1 of P. gallinaceum. Assuming a date of 6 million years ago (mya) for the divergence of the P. falciparum K1 and the P. reichenowi msp1 genes (on the basis of previous estimates for these parasite species as well as host divergence times), the most recent common ancestor of the dimorphic region of msp1 would date to approximately 27mya. Thus, the P. falciparum msp1 dimorphism is confirmed as one of the oldest polymorphisms known with the exception of self-incompatibility S genes in Solanaceae. In contrast with the major allelic dimorphism, the polymorphisms present in the relatively conserved C terminus of P. falciparum msp1 appear to have arisen since the divergence of the P. falciparum and P. reichenowi msp1 genes.