An immunoinformatics approach for the design of a multi-epitope subunit vaccine for urogenital schistosomiasis.

Discovery of T and B memory cells capable of eliciting long-term immunity against schistosomiasisis is important for people in endemic areas. Changes in schistosomes environment due to developmental cycle, induces up-regulation of Heat Shock Proteins (HSPs) which assist the parasite in coping with the hostile conditions associated with its life cycle. This study therefore focused on exploring the role of HSPs in urogenital schistosomiasis to develop new multi-epitope subunit vaccine against the disease using immunoinformatic approaches. The designed subunit vaccine was subjected to in silico antigenicity, immunogenicity, allergenicity and physicochemical parameters analysis. A 3D structure of the vaccine construct was predicted, followed by disulphide engineering for stability, codon adaptation and in silico cloning for proper expression and molecular protein-protein docking of vaccine construct in the vector against toll-like receptor 4 receptor, respectively. Consequently, a 493 amino acid multi-epitope vaccine construct of antigenicity probability of 0.91 was designed. This was predicted to be stable, non-allergenic in nature and safe for human use.

Shedding proportion of Toxoplasma gondii-like oocysts in feral cats and soil contamination in Oyo State, Nigeria.

Toxoplasmosis, a disease caused by the intracellular protozoan parasite Toxoplasma gondii, is transmitted through several hosts with cats serving as its definitive host. Oocysts are released with cat faeces into the environment (e.g. soil); an important medium in its transmission. The level of soil contamination with oocysts is an indicator of the level of on- going transmission. However, a dearth of information exists on the relationship between the presence of oocysts shedding cats and soil, and its importance in the transmission of T. gondii in Nigeria. In this study, the shedding proportion of T. gondii-like oocysts in cats and soil contamination levels were investigated in three communities in Ibadan, Nigeria. Soil (n = 204) and feral cat faecal samples (n = 14) were examined for the presence of oocysts using a modified sucrose flotation technique. Cat sera (n = 15) were also analysed for IgG antibodies to T. gondii by ELISA. T. gondii-like oocysts were identified in 21.4% (95% CI: 4.6-50.8) of the total cat faecal samples. The prevalence was 50% (95% CI: 6.7-93.3), 0% and 10% (95% CI: 0.3-44.5) in Akinyele, Laniba and Ajibode communities respectively. T. gondii IgG antibody was present in 86.7% of the screened cat sera (including the copropositive cats). The seroprevalence in cats was 75% in Akinyele, 0% Laniba and 90.9% for Ajibode community (P >0.05). Oocysts were recovered from 1.5% (95% CI: 0.50-4.23) of the soil samples screened and were identified from 3.8% (95% CI: 0.13-10.58) of the soil collected in Akinyele community. Akinyele also recorded the highest number of infected cats. Oocysts were identified in soil from dumpsites 2.6% (95% CI: 0.4-13.2) and residential areas 1.9% (95% CI: 0.5-6.8). Soil contaminated with T. gondii-like oocysts and cats shedding oocysts were found in areas with high human activities within the communities. The presence of T. gondii-like oocysts in the soil and the presence of cats that tested positive to antibodies specific to T. gondii MIC 3 Protein suggested the possibility of T. gondii transmission in these communities and places emphasis on its public health importance in a susceptible population.

Arsenicosis in bladder pathology and schistosomiasis in Eggua, Nigeria.

Background: Chronic schistosomiasis and arsenic exposure through drinking water are some of the risk factors for bladder cancer. To determine the association of schistosomiasis and arsenicosis with bladder pathologies, 122 individuals from Eggua in southwest Nigeria were recruited for this study. Methods: Prevalence of schistosomiasis was determined by urine microscopy and PCR. Total urinary arsenic concentration and arsenic levels in three different water sources in the community were assessed by flame atomic absorption spectrometry. Bladder pathologies were investigated by ultrasonography. The data collected were evaluated with chi-square (χ2) and ANOVA tests to examine the relationships among demographic factors, infection, bladder pathologies and urinary arsenic concentrations. Results: The prevalence and mean intensity of schistosomiasis were 21.3% and 20.7 eggs/10 mL urine, respectively. Arsenic concentration in two of the water sources, River Yewa (0.46 mg/L) and borehole (0.52 mg/L), were above the WHO standard (0.01 mg/L); and the mean concentration in urine samples, 1.17 mg/L, was also above the WHO standard (0.2 mg/L). There was no evidence of an association between bladder pathology and arsenicosis, or between schistosomiasis associated-bladder pathology and arsenicosis (p=0.66). Conclusions: Arsenicosis is a public health concern in the study population. At the moment no clear roles are envisaged for it in the development of bladder pathologies or urinary schistosomiasis-associated bladder pathologies in Eggua.

Metabolite profiling for biomarkers in Schistosoma haematobium infection and associated bladder pathologies.

BACKGROUND: Metabolic fingerprinting analysis can offer insights into underlying reactions in a biological system; hence it is crucial to the understanding of disease pathogenesis and could provide useful tools for discovering biomarkers. We sought to examine the urine and plasma metabolome in individuals affected by urogenital schistosomiasis and its associated-bladder pathologies. METHODOLOGY: Blood and midstream urine were obtained from volunteers who matched our inclusion criteria among residents from Eggua, southwestern Nigeria. Samples were screened by urinalysis, microscopy, PCR and ultrasonography, and categorised as advanced (urogenital schistosomiasis associated-bladder pathologies), infection-only (urogenital schistosomiasis alone) and controls (no infection and no pathology). Metabolites were extracted and data acquired with ultra high-performance liquid chromatography coupled with Thermo Q-Exactive orbitrap HRMS. Data was analysed with MetaboAnalyst, Workflow4Metabolomics, HMDB, LipidMaps and other bioinformatics tools, with univariate and multivariate statistics for metabolite selection. PRINCIPAL FINDINGS: There were low levels of host sex steroids, and high levels of several benzenoids, catechols and lipids (including ganglioside, phosphatidylcholine and phosphatidylethanolamine), in infection-only and advanced cases (FDR<0.05, VIP>2, delta>2.0). Metabolites involved in biochemical pathways related to chorismate production were abundant in controls, while those related to choline and sphingolipid metabolism were upregulated in advanced cases (FDR<0.05). Some of these human host and Schistosoma haematobium molecules, including catechol estrogens, were good markers to distinguish infection-only and advanced cases. CONCLUSIONS: Altered glycerophospholipid and sphingolipid metabolism could be key factors promoting the development of bladder pathologies and tumours during urogenital schistosomiasis.

Correction: The microbiome in urogenital schistosomiasis and induced bladder pathologies.

[This corrects the article DOI: 10.1371/journal.pntd.0005826.].

Quantitative label-free proteomic analysis of human urine to identify novel candidate protein biomarkers for schistosomiasis.

BACKGROUND: Schistosomiasis is a chronic neglected tropical disease that is characterized by continued inflammatory challenges to the exposed population and it has been established as a possible risk factor in the aetiology of bladder cancer. Improved diagnosis of schistosomiasis and its associated pathology is possible through mass spectrometry to identify biomarkers among the infected population, which will influence early detection of the disease and its subtle morbidity. METHODOLOGY: A high-throughput proteomic approach was used to analyse human urine samples for 49 volunteers from Eggua, a schistosomiasis endemic community in South-West, Nigeria. The individuals were previously screened for Schistosoma haematobium and structural bladder pathologies via microscopy and ultrasonography respectively. Samples were categorised into schistosomiasis, schistosomiasis with bladder pathology, bladder pathology, and a normal healthy control group. These samples were analysed to identify potential protein biomarkers. RESULTS: A total of 1306 proteins and 9701 unique peptides were observed in this study (FDR = 0.01). Fifty-four human proteins were found to be potential biomarkers for schistosomiasis and bladder pathologies due to schistosomiasis by label-free quantitative comparison between groups. Thirty-six (36) parasite-derived potential biomarkers were also identified, which include some existing putative schistosomiasis biomarkers that have been previously reported. Some of these proteins include Elongation factor 1 alpha, phosphopyruvate hydratase, histone H4 and heat shock proteins (HSP 60, HSP 70). CONCLUSION: These findings provide an in-depth analysis of potential schistosoma and human host protein biomarkers for diagnosis of chronic schistosomiasis caused by Schistosoma haematobium and its pathogenesis.

Acquisition, maintenance and adaptation of invasion inhibitory antibodies against Plasmodium falciparum invasion ligands involved in immune evasion.

Erythrocyte-binding antigens (EBAs) and P. falciparum reticulocyte-binding homologue proteins (PfRhs) are two important protein families that can vary in expression and utilization by P. falciparum to evade inhibitory antibodies. We evaluated antibodies at repeated time-points among individuals living in an endemic region in Nigeria over almost one year against these vaccine candidates. Antibody levels against EBA140, EBA175, EBA181, PfRh2, PfRh4, and MSP2, were measured by ELISA. We also used parasites with disrupted EBA140, EBA175 and EBA181 genes to show that all these were targets of invasion inhibitory antibodies. However, antigenic targets of inhibitory antibodies were not stable and changed substantially over time in most individuals, independent of age. Antibodies levels measured by ELISA also varied within and between individuals over time and the antibodies against EBA181, PfRh2 and MSP2 declined more rapidly in younger individuals (≤15 years) compared with older (>15). The breadth of high antibody responses over time was more influenced by age than by the frequency of infection. High antibody levels were associated with a more stable invasion inhibitory response, which could indicate that during the long process of formation of immunity, many changes not only in levels but also in functional responses are needed. This is an important finding in understanding natural immunity against malaria, which is essential for making an efficacious vaccine.

The microbiome in urogenital schistosomiasis and induced bladder pathologies.

BACKGROUND: Human schistosomiasis is a highly prevalent neglected tropical disease (NTD) caused by Schistosoma species. Research on the molecular mechanisms influencing the outcomes of bladder infection by Schistosoma haematobium is urgently needed to develop new diagnostics, therapeutics and infection prevention strategies. The objective of the research study was to determine the microbiome features and changes in urine during urogenital schistosomiasis and induced bladder pathologies. METHODOLOGY: Seventy participants from Eggua, southwestern Nigeria provided morning urine samples and were screened for urogenital schistosomiasis infection and bladder pathologies in a cross-sectional study. Highthroughput NGS sequencing was carried out, targeting the 16S V3 region. Filtered reads were processed and analyzed in a bioinformatics pipeline. PRINCIPAL FINDINGS: The study participants (36 males and 34 females, between ages 15 and 65) were categorized into four groups according to status of schistosomiasis infection and bladder pathology. Data analytics of the next-generation sequencing reads revealed that Proteobacteria and Firmicutes dominated and had influence on microbiome structure of both non-infected persons and persons with urogenital schistosomiasis. Furthermore, gender and age influenced taxa abundance independent of infection or bladder pathology. Several taxa distinguished urogenital schistosomiasis induced bladder pathologies from urogenital schistosomiasis infection alone and from healthy persons, including known immune-stimulatory taxa such as Fusobacterium, Sphingobacterium and Enterococcus. Some of these significant taxa, especially Sphingobacterium were projected as markers of infection, while several genera including potentially beneficial taxa such as Trabulsiella and Weissella, were markers of the non-infected. Finally, expected changes in protein functional categories were observed to relate to cellular maintenance and lipid metabolism. CONCLUSION: The urinary microbiome is a factor to be considered in developing biomarkers, diagnostic tools, and new treatment for urogenital schistosomiasis and induced bladder pathologies.

Antimalarial actions of Lawsonia inermis, Tithonia diversifolia and Chromolaena odorata in combination.

ETHNOPHARMACOLOGICAL RELEVANCE: Chromolaena odorata, Tithonia diversifolia and Lawsonia inermis are medicinal plants used in treating malaria in traditional medicine system. Previous studies however showed that their dichloromethane, methanol (1:1) extracts were more active against Plasmodium parasite than the aqueous extracts. AIM OF THE STUDY: To determine the in vitro and in vivo antiplasmodial activity of dichloromethane, methanol (1:1) extracts of Chromolaena odorata, Tithonia diversifolia and Lawsonia inermis in combination and evaluate their safety using acute limit toxicity test. MATERIALS AND METHODS: Dichloromethane, methanol (1:1) extracts of Chromolaena odorata, Tithonia diversifolia and Lawsonia inermis leaves were combined at ratios 1:1, 1:3, 3:1, 1:5 and 5:1 using in vitro semi-automated microdilution technique against P. falciparum Chloroquine sensitive (D6) and Chloroquine resistant (W2) strains, with chloroquine and artemisinin as controls. The in vivo antiplasmodial activity of the crude extracts was carried out singly, and in combination at the different combination ratios on Plasmodium berghei Anka infected Swiss albino mice using Peters' 4-day suppressive test. Acute toxicity test was done in mice at 5000mg/kg. RESULTS: The in vitro combination of L. inermis and T. diversifolia (1:1) extracts against P. falciparum showed the highest synergy with IC50 of 0.43±0.02µg/mL and 2.55±0.19µg/mL against D6 and W2 respectively; while the combination of C. odorata with T. diversifolia and L. inermis were antagonistic. A synergy with chemosuppression of 83.6% against P. berghei infected mice was observed in L. inermis and T. diversifolia (1:1) treated animals. In contrast to the in vitro result, combination of C. odorata with T. diversifolia and L. inermis showed some degrees of synergy in vivo. Extracts were not toxic at the concentration tested. CONCLUSION: These findings rationalized the use of these plants in combination as antimalarials in traditional medicine. However, the combination of Chromolaena odorata with other medicinal plants should be used with caution because of its possible antagonistic effect.

Persistent Transmission of Schistosomiasis in Southwest Nigeria: Contexts of Culture and Contact with Infected River Water.

Transmission of schistosomiasis is aided by human behaviour. Globally, about 800 million people are at risk of schistosomiasis infection. Data exist on biomedical understanding of the disease transmission; there is a dearth of information from the social science perspective. Hence, this study explored the social and cultural context of schistosomiasis transmission among Yewa People in Nigeria. Qualitative methods were employed with purposive sampling, using the key informant interviews and focus group discussions, among 57 participants aged 17 to 54 years. The data were content-analyzed. River water was the most reported source of water supply among others. Participants drew from the cultural milieu the use of river water for "drinking" and "swimming" as part of the continual transmission of schistosomiasis. Transmission of schistosomiasis may not be abated without behavioural change.

Developmental Regulation of Genes Encoding Universal Stress Proteins in Schistosoma mansoni.

The draft nuclear genome sequence of the snail-transmitted, dimorphic, parasitic, platyhelminth Schistosoma mansoni revealed eight genes encoding proteins that contain the Universal Stress Protein (USP) domain. Schistosoma mansoni is a causative agent of human schistosomiasis, a severe and debilitating Neglected Tropical Disease (NTD) of poverty, which is endemic in at least 76 countries. The availability of the genome sequences of Schistosoma species presents opportunities for bioinformatics and genomics analyses of associated gene families that could be targets for understanding schistosomiasis ecology, intervention, prevention and control. Proteins with the USP domain are known to provide bacteria, archaea, fungi, protists and plants with the ability to respond to diverse environmental stresses. In this research investigation, the functional annotations of the USP genes and predicted nucleotide and protein sequences were initially verified. Subsequently, sequence clusters and distinctive features of the sequences were determined. A total of twelve ligand binding sites were predicted based on alignment to the ATP-binding universal stress protein from Methanocaldococcus jannaschii. In addition, six USP sequences showed the presence of ATP-binding motif residues indicating that they may be regulated by ATP. Public domain gene expression data and RT-PCR assays confirmed that all the S. mansoni USP genes were transcribed in at least one of the developmental life cycle stages of the helminth. Six of these genes were up-regulated in the miracidium, a free-swimming stage that is critical for transmission to the snail intermediate host. It is possible that during the intra-snail stages, S. mansoni gene transcripts for universal stress proteins are low abundant and are induced to perform specialized functions triggered by environmental stressors such as oxidative stress due to hydrogen peroxide that is present in the snail hemocytes. This report serves to catalyze the formation of a network of researchers to understand the function and regulation of the universal stress proteins encoded in genomes of schistosomes and their snail intermediate hosts.

Fine specificity of anti-MSP119 antibodies and multiplicity of Plasmodium falciparum merozoite surface protein 1 types in individuals in Nigeria with sub-microscopic infection.

BACKGROUND: The absence of antibodies specific for the 19 kDa C-terminal domain of merozoite surface protein 1 (MSP119) has been associated with high-density malaria parasitaemia in African populations. The hypothesis that a high prevalence and/or level of anti-MSP119 antibodies that may inhibit erythrocyte invasion would be present in apparently healthy individuals who harbour a sub-microscopic malaria infection was tested in this study. METHODS: Plasma samples were collected from residents in a region in Nigeria hyperendemic for malaria, who had no detectable parasitaemia by microscopy. Using a competition-based enzyme-linked-immunosorbent assay with two invasion-inhibitory monoclonal antibodies (mAbs) 12.10 and 12.8, the levels and prevalence of specific antibodies were measured. The minimum multiplicity of infection was determined using PCR. The prevalence of anaemia was also measured. RESULTS: Plasma samples from 85% of individuals contained antibodies that bound to MSP119. The inhibition of mAb 12.10 binding was strongly correlated with the prevalence (Spearman correlation test, p < 0.0001) and mean titre of anti-MSP119 antibodies (Spearman correlation test, p < 0.001) in the samples. Comparing samples from individuals with multiple infection (group M) and single infection (Group S), group M contained a higher (p = 0.04) prevalence of anti-MSP119 antibodies that competed with mAb 12.10. Using a logistic regression model, it was found that the presence of antibodies competitive with mAb 12.10 was affected negatively by anaemia (p = 0.0016) and positively by the carriage of multiple parasite genotypes (p = 0.04). CONCLUSIONS: In the search for correlates of protection against malaria, which will be essential to evaluate clinical trials of malaria vaccines based on MSP1, this study examines some potential assays and the factors that need to taken into account during their evaluation, using samples from individuals naturally exposed to malaria infection.

Cellular responses to modified Plasmodium falciparum MSP119 antigens in individuals previously exposed to natural malaria infection.

BACKGROUND: MSP1 processing-inhibitory antibodies bind to epitopes on the 19 kDa C-terminal region of the Plasmodium falciparum merozoite surface protein 1 (MSP1(19)), inhibiting erythrocyte invasion. Blocking antibodies also bind to this antigen but prevent inhibitory antibodies binding, allowing invasion to proceed. Recombinant MSP1(19) had been modified previously to allow inhibitory but not blocking antibodies to continue to bind. Immunization with these modified proteins, therefore, has the potential to induce more effective protective antibodies. However, it was unclear whether the modification of MSP1(19) would affect critical T-cell responses to epitopes in this antigen. METHODS: The cellular responses to wild-type MSP1(19) and a panel of modified MSP1(19) antigens were measured using an in-vitro assay for two groups of individuals: the first were malaria-naïve and the second had been naturally exposed to Plasmodium falciparum infection. The cellular responses to the modified proteins were examined using cells from malaria-exposed infants and adults. RESULTS: Interestingly, stimulation indices (SI) for responses induced by some of the modified proteins were at least two-fold higher than those elicited by the wild-type MSP1(19). A protein with four amino acid substitutions (Glu27-->Tyr, Leu31-->Arg, Tyr34-->Ser and Glu43-->Leu) had the highest stimulation index (SI up to 360) and induced large responses in 64% of the samples that had significant cellular responses to the modified proteins. CONCLUSION: This study suggests that specific MSP1(19) variants that have been engineered to improve their antigenicity for inhibitory antibodies, retain T-cell epitopes and the ability to induce cellular responses. These proteins are candidates for the development of MSP1-based malaria vaccines.

Cytokine profiles and antibody responses to Plasmodium falciparum malaria infection in individuals living in Ibadan, southwest Nigeria.

BACKGROUND: The ability of the host immune system to efficiently clear Plasmodium falciparum parasites during a malaria infection depends on the type of immune response mounted by the host. STUDY DESIGN: In a cross-sectional study, we investigated the cellular-and antibody responses in individuals with P. falciparum infection, in an attempt to identify immunological signs indicative of the development of natural immunity against malaria in Ibadan, Nigeria. Levels of IL-10, IL-12(p70), IFN-gamma, and IgM, IgG and IgG1-4 subclasses in the serum of 36 symptomatic children with microscopically confirmed malaria parasitaemia and 54 asymptomatic controls were analysed by ELISA. RESULTS: IFN-gamma and IL-10 were significantly higher in the symptomatic children (p=0.009, p=0.025 respectively) than in the asymptomatic controls but no differences were seen for IL-12(p70). Estimated higher ratios of IFN-gamma/IL-10 and IFN-gamma/IL-12 were also observed in the symptomatic children while the asymptomatic controls had higher IL-12/IL-10 ratio. The mean concentration levels of anti-P. falciparum IgG1, IgG2, IgG3 antibodies were statistically significantly higher in the individuals >5 years of age than <5 years while anti-P. falciparum IgG3 antibodies were notably low in <5 years category. Children <5 years had higher IgM antibodies than IgG and the expression of IgG subclasses increased with age. CONCLUSION: Taken together, malaria infection is on a delicate balance of pro- and anti-inflammatory cytokines. The higher levels of IFN-gamma seen in the symptomatic children (<6 months) may be instrumental in immune-protection against malaria by limiting parasite replication. The observed variations in immunoglobulin subclass levels were age-dependent and exposure-related.

Detection of p53 codon 249 mutation in Nigerian patients with hepatocellular carcinoma using a novel evaluation of cell-free DNA.

OBJECTIVES: This case-control study was done to determine the association and prevalence of p53 codon 249 mutation using cell-free DNA in the plasma of patients with hepatocellular carcinoma (HCC) in South-Western Nigeria. METHOD: Eighty-five adults with HCC and seventy-seven age and gender matched controls without evidence of liver disease or malignancy involving any part of the body, were recruited. Plasma DNA was analyzed for p53 codon 249 by restriction fragment length polymorphism. Patient evaluation was done by means questionnaire interview, clinical examination, laboratory and radiological tests. The prevalence of the p53 codon 249 mutation was expressed as a percentage amplifiable DNA samples analyzed from HCC patients while that of controls was expressed in the same way. Fisher's exact test or the student t-test where appropriate were used to assess statistical significance of prevalence between both groups as well as comparison of some characteristics in the HCC cases between those who had codon 249 mutation and those who did not. Associations between the various parameters assessed were determined by odds ratio and significant difference was specified at p < 0.05. RESULTS: p53 codon 249 mutation was present in 6 (7.6%) of the 79 samples from the HCC patients with amplifiable plasma DNA while none (i.e. 0%) of the 73 samples with amplifiable plasma DNA from the controls had this mutation. This prevalence is significantly higher among HCC patients than controls (0.029). The mutation was also found to be significantly associated with HCC (odds ratio = 2.00; 95% C I: 1.70 - 2.35). CONCLUSION: The prevalence of the p53 codon 249 mutation from plasma DNA of hepatocellular carcinoma patients is significantly higher than among controls in South-Western Nigeria and the presence of this mutation is significantly associated with HCC in this region.

Epidemiological factors that promote the development of severe malaria anaemia in children in Ibadan.

BACKGROUND: Effective control and management of severe malaria cases depends on a clear understanding of the local epidemiological factors and specific clinical manifestations of the disease in the different endemic regions. OBJECTIVES: To determine the prevalence of severe malaria and epidemiological factors that affect the development of malaria anaemia. METHODS: A cross-sectional survey was carried out among children below 5 years of age, at the Adeoyo State Maternity Hospital, Ibadan, Nigeria. Questionnaires and case histories were taken from patients clinically diagnosed of malaria. Thus, 372 volunteers were recruited into the study from the 3131 paediatric cases that reported over the 10-week period to the out-patient department (OPD) of the hospital. 229 (61.6%) of the recruited volunteers presented with fever (>37.5 degrees C) at consultation. These had malaria parasite and PCV tests done. RESULTS: Clinical diagnosis was confirmed microscopically in 78% (290/372) for Plasmodium infection using thick film slides. Anaemia (PCV <28%) prevalence was 28.2%. Factors that contributed to the rapid progression of uncomplicated malaria to severe status included: age of the child, level of parasitaemia, careless response and attitude of parents or guardians to fever in the children; parents' preoccupation with their jobs or other healthy children and unwillingness to use available health facilities. CONCLUSION: The study underscores the need for community involved partnership for malaria control especially through health education for the home management of malaria, especially among those experiencing some form of inequity in access to healthcare.

The human immune response to Plasmodium falciparum includes both antibodies that inhibit merozoite surface protein 1 secondary processing and blocking antibodies.

Malaria merozoite surface protein 1 (MSP1) is cleaved in an essential step during erythrocyte invasion. The responses of children to natural malaria infection included antibodies that inhibit this cleavage and others that block the binding of these inhibitory antibodies. There was no correlation between the titer of the antibody to the 19-kDa fragment of MSP1 and its inhibitory activity. These findings have implications for the design of MSP1-based vaccines.