Identifying Merozoite Targets of Protective Immunity Against <i>Plasmodium falciparum</i> Malaria

The observation that individuals living in malaria endemic regions who are repeatedly infected with P. falciparum can acquire immunity, first to severe, then to uncomplicated clinical episodes of malaria, and finally to high parasite densities, provides hope that a vaccine is achievable. Immunoglobulins have been identified as a key component of naturally acquired immunity and identifying the targets and mechanisms by which this protection is achieved is a clear research priority. To date, only a small proportion of the parasite proteome has been evaluated in the context of naturally acquired immunity. This thesis was aimed at contributing to this knowledge gap by identifying novel potential antigen targets of protective antibodies and validating these in samples from Tanzanian adults. First, to identify merozoite antigens that were immunogenic, I resolved proteins extracted from P. falciparum merozoites by two-dimensional gel electrophoresis and tested these for reactivity with immunoglobulins from malaria immune adults. Immunoreactive proteins were then identified by mass-spectrometry. In complementary studies, purified P. falciparum merozoites were treated with proteolytic enzymes to release proteins localised on the surface of merozoites, which were subsequently identified by mass-spectrometry. Using stringent criteria, where I combined the data obtained from 2D-immunoblots and surface-trypsinization experiments with bioinformatics prediction (for the presence of a signal peptide and/or transmembrane domain), I narrowed down to 222 potential merozoite vaccine targets. These included known surface and/or immunogenic proteins such as the 6-cysteine proteins (Pf12, Pf38, Pf41), MSP-1, 3, 7, 9, 10, GLURP, AMA1, GAMA, MTRAP, LSA3 and RhopH3 as well as many unstudied proteins. From the set of unstudied proteins, I prioritised 27 antigens for immunoprofiling and identified 19 antigens that are targets of naturally acquired antibodies and potential novel vaccine candidates. Next, using a cohort of adults living in a village in Tanzania that experiences hyperendemic malaria transmission throughout the year, I examined antibody responses to the novel potential vaccine candidates to test whether they were correlated with protective immunity. I began by identifying a panel of antigens that were immunogenic and elicited a stable antibody response in adults. Subsequently, I identified six antigens that were individually associated with protection from clinical episodes of malaria. Individuals who became ill during the follow-up period had significantly lower levels of these antibodies compared to those who did not. These antigens were the pantothenate transporter (PfPAT), a putative amino acid transporter (PF3D7_0629500), PF3D7_0830500, PF3D7_1025300, PF3D7_1345100 and PF3D7_1401600. In addition, the breadth of antibody responses to the tested antigens was associated with protection from clinical malaria. Finally, four of these six antigens strongly correlated with protective effector functions. Antibody responses to PfPAT were strongly correlated to both the ability to fix soluble factor C1q (C1q-fixation) to merozoites as well as with their interaction with neutrophils to release reactive species (ADRB). In addition, antibody responses to the putative amino acid transporter, PF3D7_1345100 and PF3D7_1401600 were strongly associated with C1q-fixation ability. Recruitment of the soluble factor C1q onto the surface of merozoite results in lysis via the classical complement pathway. The release of reactive oxygen species by neutrophils is thought to be toxic to the intra-erythrocytic stages of the parasites and is associated with parasite clearance. This thesis shows that 19 antigens, some of which have been studied for the first time in this work, are targets of naturally acquired antibody responses. Six of these antigens appeared to be associated with protective immunity to malaria in adults and correlated strongly with immune effector mechanisms that are thought to be important for parasite clearance. These findings provide a set of antigens that warrant further evaluation for inclusion into the vaccine pre-clinical development pipeline

Prevalence and risk factors for active convulsive epilepsy in rural northeast South Africa

Rationale: Epilepsy is among the most common neurological disorders worldwide. However,there are few large, population-based studies of the prevalence and risk factors for epilepsy in southern Africa. Methods: From August 2008 to February 2009, as part of a multi-site study, we undertook a three-stage, population-based study, embedded within the Agincourt health and socio-demographic surveillance system, to estimate the prevalence and identify risk factors of active convulsiveepilepsy (ACE) in a rural South African population. Results: The crude prevalence of ACE, after adjusting for non-response and the sensitivity of the screening method, was 7.0/1,000 individuals (95%CI 6.4—7.6) with significant geographic hetero-geneity across the study area. Being male (OR = 2.3; 95%CI 1.6—3.2), family history of seizures(OR = 4.0; 95%CI 2.0—8.1), a sibling with seizures (OR = 7.0; 95%CI 1.6—31.7), problems after deliv-ery (OR = 5.9; 95%CI 1.2—24.6), and history of snoring (OR = 6.5; 95%CI 4.5—9.5) were significantlyassociated with ACE. For children, their mother’s exposure to some formal schooling was pro-tective (OR = 0.30; 95%CI 0.11—0.84) after controlling for age and sex. Human immunodeficiencyvirus was not found to be associated with ACE. Conclusions: ACE is less frequent in this part of rural South Africa than other parts of sub-SaharanAfrica. Improving obstetric services could prevent epilepsy. The relationship between snoring and ACE requires further investigation, as does the relative contribution of genetic and environmental factors to examine the increased risk in those with a family history of epilepsy

Plasmodium falciparum Merozoite Associated Armadillo Protein (PfMAAP) Is Apically Localized in Free Merozoites and Antibodies Are Associated With Reduced Risk of Malaria.

Understanding the functional role of proteins expressed by Plasmodium falciparum is an important step toward unlocking potential targets for the development of therapeutic or diagnostic interventions. The armadillo (ARM) repeat protein superfamily is associated with varied functions across the eukaryotes. Therefore, it is important to understand the role of members of this protein family in Plasmodium biology. The Plasmodium falciparum armadillo repeats only (PfARO; Pf3D7_0414900) and P. falciparum merozoite organizing proteins (PfMOP; Pf3D7_0917000) are armadillo-repeat containing proteins previously characterized in P. falciparum. Here, we describe the characterization of another ARM repeat-containing protein in P. falciparum, which we have named the P. falciparum Merozoites-Associated Armadillo repeats protein (PfMAAP). Antibodies raised to three different synthetic peptides of PfMAAP show apical staining of free merozoites and those within the mature infected schizont. We also demonstrate that the antibodies raised to the PfMAAP peptides inhibited invasion of erythrocytes by merozoites from different parasite isolates. In addition, naturally acquired human antibodies to the N- and C- termini of PfMAAP are associated with a reduced risk of malaria in a prospective cohort analysis

Prevalence and factors associated with convulsive status epilepticus in Africans with epilepsy

Objective: We conducted a community survey to estimate the prevalence and describe the features, risk factors, and consequences of convulsive status epilepticus (CSE) among people with active convulsive epilepsy (ACE) identified in a multisite survey in Africa. Methods: We obtained clinical histories of CSE and neurologic examination data among 1,196 people with ACE identified from a population of 379,166 people in 3 sites: Agincourt, South Africa; Iganga-Mayuge, Uganda; and Kilifi, Kenya. We performed serologic assessment for the presence of antibodies to parasitic infections and HIV and determined adherence to antiepileptic drugs. Consequences of CSE were assessed using a questionnaire. Logistic regression was used to identify risk factors. Results: The adjusted prevalence of CSE in ACE among the general population across the 3 sites was 2.3 per 1,000, and differed with site (p \u3c 0.0001). Over half (55%) of CSE occurred in febrile illnesses and focal seizures were present in 61%. Risk factors for CSE in ACE were neurologic impairments, acute encephalopathy, previous hospitalization, and presence of antibody titers to falciparum malaria and HIV; these differed across sites. Burns (15%), lack of education (49%), being single (77%), and unemployment (78%) were common in CSE; these differed across the 3 sites. Nine percent with and 10% without CSE died. Conclusions: CSE is common in people with ACE in Africa; most occurs with febrile illnesses, is untreated, and has focal features suggesting preventable risk factors. Effective prevention and the management of infections and neurologic impairments may reduce the burden of CSE in ACE

Plasmodium falciparum malaria parasite var gene expression is modified by host antibodies: longitudinal evidence from controlled infections of Kenyan adults with varying natural exposure.

BACKGROUND: The PfEMP1 family of Plasmodium falciparum antigens play a key role in pathogenesis of severe malaria through their insertion into the surface of parasite infected erythrocytes, and adhesion to host cells. Previous studies have suggested that parasites expressing PfEMP1 subclasses group A and DC8, associated with severe malaria, may have a growth advantage in immunologically naïve individuals. However, this idea has not been tested in longitudinal studies. METHODS: Here we assessed expression of the var genes encoding PfEMP1, in parasites sampled from volunteers with varying prior exposure to malaria, following experimental infection by sporozoites (PfSPZ). Using qPCR, we tested for associations between the expression of various var subgroups in surviving parasite populations from each volunteer and 1) the levels of participants' antibodies to infected erythrocytes before challenge infection and 2) the apparent in vivo parasite multiplication rate. RESULTS: We show that 1) expression of var genes encoding for group A and DC8-like PfEMP1 were associated with low levels of antibodies to infected erythrocytes (αIE) before challenge, and 2) expression of a DC8-like CIDRα1.1 domain was associated with higher apparent parasite multiplication rate in a manner that was independent of levels of prior antibodies to infected erythrocytes. CONCLUSIONS: This study provides insight into the role of antibodies to infected erythrocytes surface antigens in the development of naturally acquired immunity and may help explain why specific PfEMP1 variants may be associated with severe malaria. TRIAL REGISTRATION: Pan African Clinical Trial Registry: PACTR201211000433272 . Date of registration: 10th October 2012

Sequential Vaccination With Heterologous Acinetobacter baumannii Strains Induces Broadly Reactive Antibody Responses

Antibody therapy may be an alternative treatment option for infections caused by the multi-drug resistant (MDR) bacterium Acinetobacter baumannii. As A. baumannii has multiple capsular serotypes, a universal antibody therapy would need to target conserved protein antigens rather than the capsular polysaccharides. We have immunized mice with single or multiple A. baumannii strains to induce antibody responses to protein antigens, and then assessed whether these responses provide cross-protection against a collection of genetically diverse clinical A. baumannii isolates. Immunized mice developed antibody responses to multiple protein antigens. Flow cytometry IgG binding assays and immunoblots demonstrated improved recognition of both homologous and heterologous clinical strains in sera from mice immunized with multiple strains compared to a single strain. The capsule partially inhibited bacterial recognition by IgG and the promotion of phagocytosis by human neutrophils. However, after immunization with multiple strains, serum antibodies to protein antigens promoted neutrophil phagocytosis of heterologous A. baumannii strains. In an infection model, mice immunized with multiple strains had lower bacterial counts in the spleen and liver following challenge with a heterologous strain. These data demonstrate that antibodies targeting protein antigens can improve immune recognition and protection against diverse A. baumannii strains, providing support for their use as an antibody therapy

KILchip v1.0: A Novel Plasmodium falciparum Merozoite Protein Microarray to Facilitate Malaria Vaccine Candidate Prioritization

Passive transfer studies in humans clearly demonstrated the protective role of IgG antibodies against malaria. Identifying the precise parasite antigens that mediate immunity is essential for vaccine design, but has proved difficult. Completion of the Plasmodium falciparum genome revealed thousands of potential vaccine candidates, but a significant bottleneck remains in their validation and prioritization for further evaluation in clinical trials. Focusing initially on the Plasmodium falciparum merozoite proteome, we used peer-reviewed publications, multiple proteomic and bioinformatic approaches, to select and prioritize potential immune targets. We expressed 109 P. falciparum recombinant proteins, the majority of which were obtained using a mammalian expression system that has been shown to produce biologically functional extracellular proteins, and used them to create KILchip v1.0: a novel protein microarray to facilitate high-throughput multiplexed antibody detection from individual samples.The microarray assay was highly specific; antibodies against P. falciparum proteins were detected exclusively in sera from malaria-exposed but not malaria-naïve individuals. The intensity of antibody reactivity varied as expected from strong to weak across well-studied antigens such as AMA1 and RH5 (Kruskal–Wallis H test for trend: p &lt; 0.0001). The inter-assay and intra-assay variability was minimal, with reproducible results obtained in re-assays using the same chip over a duration of 3 months. Antibodies quantified using the multiplexed format in KILchip v1.0 were highly correlated with those measured in the gold-standard monoplex ELISA [median (range) Spearman's R of 0.84 (0.65–0.95)]. KILchip v1.0 is a robust, scalable and adaptable protein microarray that has broad applicability to studies of naturally acquired immunity against malaria by providing a standardized tool for the detection of antibody correlates of protection. It will facilitate rapid high-throughput validation and prioritization of potential Plasmodium falciparum merozoite-stage antigens paving the way for urgently needed clinical trials for the next generation of malaria vaccines

The ratio of monocytes to lymphocytes in peripheral blood correlates with increased susceptibility to clinical malaria in Kenyan children.

BACKGROUND: Plasmodium falciparum malaria remains a major cause of illness and death in sub-Saharan Africa. Young children bear the brunt of the disease and though older children and adults suffer relatively fewer clinical attacks, they remain susceptible to asymptomatic P. falciparum infection. A better understanding of the host factors associated with immunity to clinical malaria and the ability to sustain asymptomatic P. falciparum infection will aid the development of improved strategies for disease prevention. METHODS AND FINDINGS: Here we investigate whether full differential blood counts can predict susceptibility to clinical malaria among Kenyan children sampled at five annual cross-sectional surveys. We find that the ratio of monocytes to lymphocytes, measured in peripheral blood at the time of survey, directly correlates with risk of clinical malaria during follow-up. This association is evident among children with asymptomatic P. falciparum infection at the time the cell counts are measured (Hazard ratio (HR)  =  2.7 (95% CI 1.42, 5.01, P  =  0.002) but not in those without detectable parasitaemia (HR  =  1.0 (95% CI 0.74, 1.42, P  =  0.9). CONCLUSIONS: We propose that the monocyte to lymphocyte ratio, which is easily derived from routine full differential blood counts, reflects an individual's capacity to mount an effective immune response to P. falciparum infection

KILchip v1.0: A Novel Plasmodium falciparum Merozoite Protein Microarray to Facilitate Malaria Vaccine Candidate Prioritization.

Passive transfer studies in humans clearly demonstrated the protective role of IgG antibodies against malaria. Identifying the precise parasite antigens that mediate immunity is essential for vaccine design, but has proved difficult. Completion of the Plasmodium falciparum genome revealed thousands of potential vaccine candidates, but a significant bottleneck remains in their validation and prioritization for further evaluation in clinical trials. Focusing initially on the Plasmodium falciparum merozoite proteome, we used peer-reviewed publications, multiple proteomic and bioinformatic approaches, to select and prioritize potential immune targets. We expressed 109 P. falciparum recombinant proteins, the majority of which were obtained using a mammalian expression system that has been shown to produce biologically functional extracellular proteins, and used them to create KILchip v1.0: a novel protein microarray to facilitate high-throughput multiplexed antibody detection from individual samples. The microarray assay was highly specific; antibodies against P. falciparum proteins were detected exclusively in sera from malaria-exposed but not malaria-naïve individuals. The intensity of antibody reactivity varied as expected from strong to weak across well-studied antigens such as AMA1 and RH5 (Kruskal-Wallis H test for trend: p < 0.0001). The inter-assay and intra-assay variability was minimal, with reproducible results obtained in re-assays using the same chip over a duration of 3 months. Antibodies quantified using the multiplexed format in KILchip v1.0 were highly correlated with those measured in the gold-standard monoplex ELISA [median (range) Spearman's R of 0.84 (0.65-0.95)]. KILchip v1.0 is a robust, scalable and adaptable protein microarray that has broad applicability to studies of naturally acquired immunity against malaria by providing a standardized tool for the detection of antibody correlates of protection. It will facilitate rapid high-throughput validation and prioritization of potential Plasmodium falciparum merozoite-stage antigens paving the way for urgently needed clinical trials for the next generation of malaria vaccines

Development and validation of a diagnostic aid for convulsive epilepsy in sub-Saharan Africa: a retrospective case-control study

Background: Identification of convulsive epilepsy in sub-Saharan Africa relies on access to resources that are often unavailable. Infrastructure and resource requirements can further complicate case verification. Using machine-learning techniques, we have developed and tested a region-specific questionnaire panel and predictive model to identify people who have had a convulsive seizure. These findings have been implemented into a free app for health-care workers in Kenya, Uganda, Ghana, Tanzania, and South Africa. Methods: In this retrospective case-control study, we used data from the Studies of the Epidemiology of Epilepsy in Demographic Sites in Kenya, Uganda, Ghana, Tanzania, and South Africa. We randomly split these individuals using a 7:3 ratio into a training dataset and a validation dataset. We used information gain and correlation-based feature selection to identify eight binary features to predict convulsive seizures. We then assessed several machine-learning algorithms to create a multivariate prediction model. We validated the best-performing model with the internal dataset and a prospectively collected external-validation dataset. We additionally evaluated a leave-one-site-out model (LOSO), in which the model was trained on data from all sites except one that, in turn, formed the validation dataset. We used these features to develop a questionnaire-based predictive panel that we implemented into a multilingual app (the Epilepsy Diagnostic Companion) for health-care workers in each geographical region. Findings: We analysed epilepsy-specific data from 4097 people, of whom 1985 (48·5%) had convulsive epilepsy, and 2112 were controls. From 170 clinical variables, we initially identified 20 candidate predictor features. Eight features were removed, six because of negligible information gain and two following review by a panel of qualified neurologists. Correlation-based feature selection identified eight variables that demonstrated predictive value; all were associated with an increased risk of an epileptic convulsion except one. The logistic regression, support vector, and naive Bayes models performed similarly, outperforming the decision-tree model. We chose the logistic regression model for its interpretability and implementability. The area under the receiver operator curve (AUC) was 0·92 (95% CI 0·91–0·94, sensitivity 85·0%, specificity 93·7%) in the internal-validation dataset and 0·95 (0·92–0·98, sensitivity 97·5%, specificity 82·4%) in the external-validation dataset. Similar results were observed for the LOSO model (AUC 0·94, 0·93–0·96, sensitivity 88·2%, specificity 95·3%). Interpretation: On the basis of these findings, we developed the Epilepsy Diagnostic Companion as a predictive model and app offering a validated culture-specific and region-specific solution to confirm the diagnosis of a convulsive epileptic seizure in people with suspected epilepsy. The questionnaire panel is simple and accessible for health-care workers without specialist knowledge to administer. This tool can be iteratively updated and could lead to earlier, more accurate diagnosis of seizures and improve care for people with epilepsy