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

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

Exposure to parasitic infections determines features and phenotypes of active convulsive epilepsy in Africa

Background: Epilepsy affects 70 million people worldwide, 80% of whom are in low-and-middle income countries (LMICs). Infections of the central nervous system (CNS) contribute considerably to the burden of epilepsy in LMICs, but the nature and presentation of epilepsy following these infections is not fully understood. We examined if epilepsy foutcomes are associated with the exposure to parasitic infections. Methods: This was a case-comparison study nested in a cross-sectional survey of people with active convulsive epilepsy, with cases as those exposed to parasitic infections, and comparison as those unexposed. Associations of exposure to parasites with clinical and electroencephalographic features of epilepsy were done using a modified mixed effects Poisson regression model across five sites in Africa. Multiplicative and additive scale (RERI) interactions were explored to determine the effect of co-infections on epilepsy features. Population attributable fractions (PAF) were calculated to determine the proportion of severe clinical and electroencephalographic features of epilepsy attributable to CNS infections. Results: A total of 997 participants with active convulsive epilepsy from the five African sites were analyzed, 51% of whom were males. Exposure to parasitic infections was associated with more frequent seizures in adult epilepsy (relative risk (RR)=2.58, 95% confidence interval (95%CI):1.71-3.89). In children, exposure to any parasite was associated with convulsive status epilepticus (RR=4.68, (95%CI: 3.79-5.78), intellectual disabilities (RR=2.13, 95%CI: 1.35-3.34) and neurological deficits (RR=1.92, 95%CI: 1.42-2.61). Toxoplasma gondii and Onchocerca volvulus interacted synergistically to increase the risk of status epilepticus (RERI=0.91, 95%CI=0.48-1.35) in the data pooled across the sites. Exposure to parasitic infections contributed to 30% of severe features of epilepsy as shown by PAF. Conclusions: Parasitic infections may determine features and phenotypes of epilepsy through synergistic or antagonistic interactions, which can be different in children and adults. Interventions to control or manage infections may reduce complications and improve prognosis in epilepsy

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

Changes in Serological Immunology Measures in UK and Kenyan Adults Post-controlled Human Malaria Infection.

Background: The timing of infection is closely determined in controlled human malaria infection (CHMI) studies, and as such they provide a unique opportunity to dissect changes in immunological responses before and after a single infection. The first Kenyan Challenge Study (KCS) (Pan African Clinical Trial Registry: PACTR20121100033272) was performed in 2013 with the aim of establishing the CHMI model in Kenya. This study used aseptic, cryopreserved, attenuated Plasmodium falciparum sporozoites administered by needle and syringe (PfSPZ Challenge) and was the first to evaluate parasite dynamics post-CHMI in individuals with varying degrees of prior exposure to malaria. Methods: We describe detailed serological and functional immunological responses pre- and post-CHMI for participants in the KCS and compare these with those from malaria-naïve UK volunteers who also underwent CHMI (VAC049) (ClinicalTrials.gov NCT01465048) using PfSPZ Challenge. We assessed antibody responses to three key blood-stage merozoite antigens [merozoite surface protein 1 (MSP1), apical membrane protein 1 (AMA1), and reticulocyte-binding protein homolog 5 (RH5)] and functional activity using two candidate measures of anti-merozoite immunity; the growth inhibition activity (GIA) assay and the antibody-dependent respiratory burst activity (ADRB) assay. Results:Clear serological differences were observed pre- and post-CHMI by ELISA between malaria-naïve UK volunteers in VAC049, and Kenyan volunteers who had prior malaria exposure. Antibodies to AMA1 and schizont extract correlated with parasite multiplication rate (PMR) post-CHMI in KCS. Serum from volunteer 110 in KCS, who demonstrated a dramatically reduced PMR in vivo, had no in vitro GIA prior to CHMI but the highest level of ADRB activity. A significant difference in ADRB activity was seen between KCS volunteers with minimal and definite prior exposure to malaria and significant increases were seen in ADRB activity post-CHMI in Kenyan volunteers. Quinine and atovaquone/proguanil, previously assumed to be removed by IgG purification, were identified as likely giving rise to aberrantly high in vitro GIA results. Conclusions: The ADRB activity assay is a promising functional assay that warrants further investigation as a measure of prior exposure to malaria and predictor of control of parasite growth. The CHMI model can be used to evaluate potential measures of naturally-acquired immunity to malaria

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

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