infection is associated with elevated circulating biomarkers of brain injury and endothelial activation.

Introduction: Malaria remains a major public health concern with substantial morbidity and mortality worldwide. In Malaysia, the emergence of has led to a surge in zoonotic malaria cases and deaths in recent years. Signs of cerebral involvement have been observed in a non-comatose, fatal case of severe knowlesi infection, but the potential impact of this malaria species on the brain remains underexplored.

Sterile protection against malaria by repeated blood stage infection in the monkey model.

The malaria parasite remains a major global public health challenge, and no vaccine is approved for use in humans. Here, we assessed whether strain-transcendent immunity can be achieved by repeated infection in monkeys. Sterile immunity was achieved after two homologous infections, whereas subsequent heterologous challenge provided only partial protection.

Avidity of anti-malarial antibodies inversely related to transmission intensity at three sites in Uganda.

Background: People living in malaria endemic areas acquire protection from severe malaria quickly, but protection from clinical disease and control of parasitaemia is acquired only after many years of repeated infections. Antibodies play a central role in protection from clinical disease; however, protective antibodies are slow to develop. This study sought to investigate the influence of Plasmodium falciparum exposure on the acquisition of high-avidity antibodies to P.

Comparison of allele frequencies of Plasmodium falciparum merozoite antigens in malaria infections sampled in different years in a Kenyan population.

Background: Plasmodium falciparum merozoite antigens elicit antibody responses in malaria-endemic populations, some of which are clinically protective, which is one of the reasons why merozoite antigens are the focus of malaria vaccine development efforts. Polymorphisms in several merozoite antigen-encoding genes are thought to arise as a result of selection by the human immune system.

Methods: The allele frequency distribution of 15 merozoite antigens over a two-year period, 2007 and 2008, was examined in parasites obtained from children with uncomplicated malaria.

Novel serologic biomarkers provide accurate estimates of recent Plasmodium falciparum exposure for individuals and communities.

Tools to reliably measure Plasmodium falciparum (Pf) exposure in individuals and communities are needed to guide and evaluate malaria control interventions. Serologic assays can potentially produce precise exposure estimates at low cost; however, current approaches based on responses to a few characterized antigens are not designed to estimate exposure in individuals. Pf-specific antibody responses differ by antigen, suggesting that selection of antigens with defined kinetic profiles will improve estimates of Pf exposure.

The effect of daily co-trimoxazole prophylaxis on natural development of antibody-mediated immunity against P. falciparum malaria infection in HIV-exposed uninfected Malawian children.

Background And Objectives: Co-trimoxazole prophylaxis, currently recommended in HIV-exposed, uninfected (HEU) children as protection against opportunistic infections, also has some anti-malarial efficacy. We determined whether daily co-trimoxazole prophylaxis affects the natural development of antibody-mediated immunity to blood-stage Plasmodium falciparum malaria infection.

Methods: Using an enzyme-linked immunosorbent assay, we measured antibodies to 8 Plasmodium falciparum antigens (AMA-1, MSP-119, MSP-3, PfSE, EBA-175RII, GLURP R0, GLURP R2 and CSP) in serum samples from 33 HEU children and 31 HIV-unexposed, uninfected (HUU) children, collected at 6, 12 and 18 months of age.

Human antibodies fix complement to inhibit Plasmodium falciparum invasion of erythrocytes and are associated with protection against malaria.

Antibodies play major roles in immunity to malaria; however, a limited understanding of mechanisms mediating protection is a major barrier to vaccine development. We have demonstrated that acquired human anti-malarial antibodies promote complement deposition on the merozoite to mediate inhibition of erythrocyte invasion through C1q fixation and activation of the classical complement pathway. Antibody-mediated complement-dependent (Ab-C') inhibition was the predominant invasion-inhibitory activity of human antibodies; most antibodies were non-inhibitory without complement.

The MSPDBL2 codon 591 polymorphism is associated with lumefantrine in vitro drug responses in Plasmodium falciparum isolates from Kilifi, Kenya.

The mechanisms of drug resistance development in the Plasmodium falciparum parasite to lumefantrine (LUM), commonly used in combination with artemisinin, are still unclear. We assessed the polymorphisms of Pfmspdbl2 for associations with LUM activity in a Kenyan population. MSPDBL2 codon 591S was associated with reduced susceptibility to LUM (P = 0.

Analysis of antibodies to newly described Plasmodium falciparum merozoite antigens supports MSPDBL2 as a predicted target of naturally acquired immunity.

Prospective studies continue to identify malaria parasite genes with particular patterns of polymorphism which indicate they may be under immune selection, and the encoded proteins require investigation. Sixteen new recombinant protein reagents were designed to characterize three such polymorphic proteins expressed in Plasmodium falciparum schizonts and merozoites: MSPDBL1 (also termed MSP3.4) and MSPDBL2 (MSP3.

Population genomic scan for candidate signatures of balancing selection to guide antigen characterization in malaria parasites.

Acquired immunity in vertebrates maintains polymorphisms in endemic pathogens, leading to identifiable signatures of balancing selection. To comprehensively survey for genes under such selection in the human malaria parasite Plasmodium falciparum, we generated paired-end short-read sequences of parasites in clinical isolates from an endemic Gambian population, which were mapped to the 3D7 strain reference genome to yield high-quality genome-wide coding sequence data for 65 isolates. A minority of genes did not map reliably, including the hypervariable var, rifin, and stevor families, but 5,056 genes (90.

The breadth, but not the magnitude, of circulating memory B cell responses to P. falciparum increases with age/exposure in an area of low transmission.

Background: Malaria caused by Plasmodium falciparum remains a major cause of death in sub-Saharan Africa. Immunity against symptoms of malaria requires repeated exposure, suggesting either that the parasite is poorly immunogenic or that the development of effective immune responses to malaria may be impaired.

Methods: We carried out two age-stratified cross-sectional surveys of anti-malarial humoral immune responses in a Gambian village where P.

A polyvalent hybrid protein elicits antibodies against the diverse allelic types of block 2 in Plasmodium falciparum merozoite surface protein 1.

Merozoite surface protein 1 (MSP1) of Plasmodium falciparum has been implicated as an important target of acquired immunity, and candidate components for a vaccine include polymorphic epitopes in the N-terminal polymorphic block 2 region. We designed a polyvalent hybrid recombinant protein incorporating sequences of the three major allelic types of block 2 together with a composite repeat sequence of one of the types and N-terminal flanking T cell epitopes, and compared this with a series of recombinant proteins containing modular sub-components and similarly expressed in Escherichia coli. Immunogenicity of the full polyvalent hybrid protein was tested in both mice and rabbits, and comparative immunogenicity studies of the sub-component modules were performed in mice.

Allelic diversity and naturally acquired allele-specific antibody responses to Plasmodium falciparum apical membrane antigen 1 in Kenya.

Although Plasmodium falciparum apical membrane antigen 1 (AMA1) is a leading malaria vaccine candidate, extensive allelic diversity may compromise its vaccine potential. We have previously shown that naturally acquired antibodies to AMA1 were associated with protection from clinical malaria in this Kenyan population. To assess the impact of allelic diversity on naturally acquired immunity, we first sequenced the ectodomain-encoding region of P.

Isolation of viable Plasmodium falciparum merozoites to define erythrocyte invasion events and advance vaccine and drug development.

During blood-stage infection by Plasmodium falciparum, merozoites invade RBCs. Currently there is limited knowledge of cellular and molecular invasion events, and no established assays are available to readily measure and quantify invasion-inhibitory antibodies or compounds for vaccine and drug studies. We report the isolation of viable merozoites that retain their invasive capacity, at high purity and yield, purified by filtration of highly synchronous populations of schizonts.

Allele frequency-based and polymorphism-versus-divergence indices of balancing selection in a new filtered set of polymorphic genes in Plasmodium falciparum.

Signatures of balancing selection operating on specific gene loci in endemic pathogens can identify candidate targets of naturally acquired immunity. In malaria parasites, several leading vaccine candidates convincingly show such signatures when subjected to several tests of neutrality, but the discovery of new targets affected by selection to a similar extent has been slow. A small minority of all genes are under such selection, as indicated by a recent study of 26 Plasmodium falciparum merozoite-stage genes that were not previously prioritized as vaccine candidates, of which only one (locus PF10_0348) showed a strong signature.

Heritability of antibody isotype and subclass responses to Plasmodium falciparum antigens.

Background: It is important to understand the extent to which genetic factors regulate acquired immunity to common infections. A classical twin study design is useful to estimate the heritable component of variation in measurable immune parameters.

Methodology/principal Findings: This study assessed the relative heritability of different plasma antibody isotypes and subclasses (IgG1, IgG2, IgG3, IgG4, IgM, IgA and IgE) naturally acquired to P.

Gene copy number variation throughout the Plasmodium falciparum genome.

Background: Gene copy number variation (CNV) is responsible for several important phenotypes of the malaria parasite Plasmodium falciparum, including drug resistance, loss of infected erythrocyte cytoadherence and alteration of receptor usage for erythrocyte invasion. Despite the known effects of CNV, little is known about its extent throughout the genome.

Results: We performed a whole-genome survey of CNV genes in P.

Prospective identification of malaria parasite genes under balancing selection.

Background: Endemic human pathogens are subject to strong immune selection, and interrogation of pathogen genome variation for signatures of balancing selection can identify important target antigens. Several major antigen genes in the malaria parasite Plasmodium falciparum have shown such signatures in polymorphism-versus-divergence indices (comparing with the chimpanzee parasite P. reichenowi), and in allele frequency based indices.

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.

Duration of naturally acquired antibody responses to blood-stage Plasmodium falciparum is age dependent and antigen specific.

Naturally acquired antibody responses provide partial protection from clinical malaria, and blood-stage parasite vaccines under development aim to prime such responses. To investigate the determinants of antibody response longevity, immunoglobulin G (IgG) antibodies to several blood-stage vaccine candidate antigens in the sera of two cohorts of children of up to 6 years of age during the dry seasons of 2003 and 2004 in The Gambia were examined. The first cohort showed that most antibodies were lost within less than 4 months of the first sampling if a persistent infection was not present, so the study of the second-year cohort involved collecting samples from individuals every 2 weeks over a 3-month period.

Progress and challenges towards the development of malaria vaccines.

The promise afforded by attenuated sporozoite vaccines in the 1970s led many researchers to believe that an efficacious malaria vaccine was an attainable medium-term goal. Over 30 years later, no licensed vaccine is currently available for public health intervention. This is despite global expenditure on research and development for malaria vaccines that is estimated to have increased from $US42 million in 1999 to $US84 million in 2004.

Haemoglobin C and S role in acquired immunity against Plasmodium falciparum malaria.

A recently proposed mechanism of protection for haemoglobin C (HbC; beta6Glu-->Lys) links an abnormal display of PfEMP1, an antigen involved in malaria pathogenesis, on the surface of HbC infected erythrocytes together with the observation of reduced cytoadhesion of parasitized erythrocytes and impaired rosetting in vitro. We investigated the impact of this hypothesis on the development of acquired immunity against Plasmodium falciparum variant surface antigens (VSA) encoding PfEMP1 in HbC in comparison with HbA and HbS carriers of Burkina Faso. We measured: i) total IgG against a single VSA, A4U, and against a panel of VSA from severe malaria cases in human sera from urban and rural areas of Burkina Faso of different haemoglobin genotypes (CC, AC, AS, SC, SS); ii) total IgG against recombinant proteins of P.

Profiling the antibody immune response against blood stage malaria vaccine candidates.

Background: The complexity and diversity of the antibody immune response to the antigen repertoire of a pathogen has long been appreciated. Although it has been recognized that the detection of antibodies against multiple antigens dramatically improves the clinical sensitivity and specificity of diagnostic assays, the prognostic value of serum reactivity profiles against multiple microbial antigens in protection has not been investigated.

Methods: Using malaria as a model we investigated whether antigen reactivity profiles in serum of children with different levels of clinical immunity to Plasmodium falciparum malaria correlated with protection.

Plasmodium falciparum merozoite surface protein 3 is a target of allele-specific immunity and alleles are maintained by natural selection.

Background: Plasmodium falciparum merozoite surface protein (MSP) 3 is an asexual blood-stage malaria vaccine candidate antigen. Sequence polymorphisms divide alleles into 2 major types, but the adaptive and immunological significance of the types has not been defined.

Methods: One hundred one msp3 allele sequences were sampled from 2 populations living in areas where malaria is endemic and were analyzed for evidence of natural selection.