Falciparum Apical Membrane Antigen Research Articles

Genetic structure of apical membrane antigen-1 in Plasmodium falciparum isolates from Pakistan.

Plasmodium falciparum apical membrane antigen-1 (PfAMA-1) is a major candidate for the blood-stage malaria vaccine. Genetic polymorphisms of global pfama-1suggest that the genetic diversity of the gene can disturb effective vaccine development targeting this antigen. This study was conducted to explore the genetic diversity and gene structure of pfama-1 among P. falciparum isolates collected in the Khyber Pakhtunkhwa (KP) province of Pakistan. A total of 19 full-length pfama-1 sequences were obtained from KP-Pakistan P. falciparum isolates, and genetic polymorphism and natural selection were investigated. KP-Pakistan pfama-1 exhibited genetic diversity, wherein 58 amino acid changes were identified, most of which were located in ectodomains, and domains I, II, and III. The amino acid changes commonly found in the ectodomain of global pfama-1 were also detected in KP-Pakistan pfama-1. Interestingly, 13 novel amino acid changes not reported in the global population were identified in KP-Pakistan pfama-1. KP-Pakistan pfama-1 shared similar levels of genetic diversity with global pfama-1. Evidence of natural selection and recombination events were also detected in KP-Pakistan pfama-1.

Genetic diversity and natural selection of apical membrane antigen-1 (ama-1) in Cameroonian Plasmodium falciparum isolates

Antigenic variation associated with genetic diversity in global Plasmodium falciparum apical membrane antigen-1 (PfAMA-1) is a major impediment to designing an effective malaria vaccine. Here, we report the first study on genetic diversity and natural selection of the Pfama-1 gene in P. falciparum isolates from Cameroon. A total of 328 P. falciparum positive samples collected during 2016 and 2019 from five localities of Cameroon were analysed. The ectodomain coding fragment of Pfama-1 gene was amplified for polymorphism profiling and natural selection analysis. A total of 108 distinct haplotypes were found in 203 P. falciparum isolates with considerable nucleotide diversity (π = 0.016) and haplotype diversity (Hd = 0.976). Most amino acid substitutions detected were scattered in ectodomain-I and few specific mutations viz P145L, K148Q, K462I, L463F, N471K, S482L, E537G, K546R and I547F were seen only in Cameroonian isolates. A tendency of natural selection towards positive diversifying selection was observed (Taj-D = 2.058). Five positively selected codon sites (P145L, S283L, Q308E/K, P330S and I547F) were identified, which overlapped with predicted B-cell epitopes and red blood cell (RBC) binding sites, suggesting their potential implication in host immune pressure and parasite-RBC binding complex modulation. The Cameroonian P. falciparum populations indicated a moderate level of genetic differentiation when compared with global sequences, with few exceptions from Vietnam and Venezuela. Our findings provide baseline data on existing Pfama-1 gene polymorphisms in Cameroonian field isolates, which will be useful information for malaria vaccine design.

Dynamics of IgG antibody response against Plasmodium antigens among Nigerian infants and young children.

Plasmodium falciparum malaria is a leading cause of child mortality in Nigeria. Neonates are born with maternal antibodies from placental transfer which may protect against malaria infection in the first months of life. The IgG dynamics of the transition from passively transferred antimalarial antibodies to actively acquired IgG from natural exposure have not been well elucidated. Blood samples collected during a 2018 Nigeria nationwide HIV/AIDS household survey were available for 9,443 children under 5 years of age, with a subset of infants under 2 months of age having maternal samples available (n=41). Samples were assayed for the P. falciparum HRP2 antigen and anti-malarial IgG antibodies. LOESS regression examined the dynamics in IgG response in the first 5 years of life. Correlation with maternal IgG levels was assessed for mother/child pairs. Consistent decreases were observed in median IgG levels against all Plasmodium spp. antigen targets for the first months of life. At a population level, P. falciparum apical membrane antigen-1 (AMA1) and merozoite surface protein-1 19kD (PfMSP1) IgG decreased during the first 12 months of life before reaching a nadir, whereas IgGs to other targets only declined for the first 4 months of life. Seropositivity showed a similar decline with the lowest seropositivity against AMA1 and PfMSP1 at 10-12 months, though remaining above 50% during the first 2 years of life in higher transmission areas. No protective association was observed between IgG positivity and P. falciparum infection in infants. Maternal antibody levels showed a strong positive correlation with infant antibody levels for all P. falciparum antigens from birth to 2 months of age, but this correlation was lost by 6 months of age. Maternally transferred anti-malarial IgG antibodies rapidly decline during the first 6 months of life, with variations among specific antigens and malaria transmission intensity. From 3-23 months of age, there was a wide range in IgG levels for the blood-stage antigens indicating high individual variation in antibody production as children are infected with malaria. Non-falciparum species-specific antigens showed similar patterns in waning immunity and correlation with paired mother's IgG levels compared to P. falciparum antigens.

Identification of factors associated with residual malaria transmission using school-based serological surveys in settings pursuing elimination

BackgroundTargeted research on residual malaria transmission is important to improve strategies in settings pursuing elimination, where transmission reductions prove challenging. This study aimed to detect and characterize spatial heterogeneity and factors associated with Plasmodium falciparum infections and exposure, P. falciparum apical membrane antigen 1 (PfAMA1) antibody (Ab) response, in the Central Highlands of Madagascar (CHL).MethodsFrom May to July 2014, a cross-sectional school-based survey was carried out in 182 fokontany (villages) within 7 health districts of the CHL. Rapid diagnostic tests (RDTs) and a bead-based immunoassay including PfAMA1 antigen biomarker were used to estimate malaria prevalence and seroprevalence, respectively. Local Moran’s I index was used to detect spatial “hotspots”. Remotely sensed environmental data—temperature, vegetation indices, land covers, and elevation—were used in multivariable mixed-effects logistic regression models to characterize factors associated with malaria infection and cumulative exposure.ResultsAmong 6,293 school-children ages 2–14 years surveyed, RDT prevalence was low at 0.8% (95% CI 0.6–1.1%), while PfAMA1 Ab seroprevalence was 7.0% (95% CI 6.4–7.7%). Hotspots of PfAMA1 Ab seroprevalence were observed in two districts (Ankazobe and Mandoto). Seroprevalence increased for children living > 5 km from a health centre (adjusted odds ratio (OR) = 1.6, 95% CI 1.2–2.2), and for those experiencing a fever episode in the previous 2 weeks (OR 1.7, 95% CI 1.2–2.4), but decreased at higher elevation (for each 100-m increase, OR = 0.7, 95% CI 0.6–0.8). A clear age pattern was observed whereby children 9–10 years old had an OR of 1.8 (95% CI 1.2–2.4), children 11–12 years an OR of 3.7 (95% CI 2.8–5.0), and children 13–14 years an OR of 5.7 (95% CI 4.0–8.0) for seropositivity, compared with younger children (2–8 years).ConclusionThe use of serology in this study provided a better understanding of malaria hotspots and associated factors, revealing a pattern of higher transmission linked to geographical barriers in health care access. The integration of antibody-assays into existing surveillance activities could improve exposure assessment, and may help to monitor the effectiveness of malaria control efforts and adapt elimination interventions.

Protein Kinase A Is Essential for Invasion of Plasmodium falciparum into Human Erythrocytes.

Understanding the mechanisms behind host cell invasion by Plasmodium falciparum remains a major hurdle to developing antimalarial therapeutics that target the asexual cycle and the symptomatic stage of malaria. Host cell entry is enabled by a multitude of precisely timed and tightly regulated receptor-ligand interactions. Cyclic nucleotide signaling has been implicated in regulating parasite invasion, and an important downstream effector of the cAMP-signaling pathway is protein kinase A (PKA), a cAMP-dependent protein kinase. There is increasing evidence that P. falciparum PKA (PfPKA) is responsible for phosphorylation of the cytoplasmic domain of P. falciparum apical membrane antigen 1 (PfAMA1) at Ser610, a cAMP-dependent event that is crucial for successful parasite invasion. In the present study, CRISPR-Cas9 and conditional gene deletion (dimerizable cre) technologies were implemented to generate a P. falciparum parasite line in which expression of the catalytic subunit of PfPKA (PfPKAc) is under conditional control, demonstrating highly efficient dimerizable Cre recombinase (DiCre)-mediated gene excision and complete knockdown of protein expression. Parasites lacking PfPKAc show severely reduced growth after one intraerythrocytic growth cycle and are deficient in host cell invasion, as highlighted by live-imaging experiments. Furthermore, PfPKAc-deficient parasites are unable to phosphorylate PfAMA1 at Ser610. This work not only identifies an essential role for PfPKAc in the P. falciparum asexual life cycle but also confirms that PfPKAc is the kinase responsible for phosphorylating PfAMA1 Ser610.IMPORTANCE Malaria continues to present a major global health burden, particularly in low-resource countries. Plasmodium falciparum, the parasite responsible for the most severe form of malaria, causes disease through rapid and repeated rounds of invasion and replication within red blood cells. Invasion into red blood cells is essential for P. falciparum survival, and the molecular events mediating this process have gained much attention as potential therapeutic targets. With no effective vaccine available, and with the emergence of resistance to antimalarials, there is an urgent need for the development of new therapeutics. Our research has used genetic techniques to provide evidence of an essential protein kinase involved in P. falciparum invasion. Our work adds to the current understanding of parasite signaling processes required for invasion, highlighting PKA as a potential drug target to inhibit invasion for the treatment of malaria.

Natural selection and genetic diversity of domain I of Plasmodium falciparum apical membrane antigen-1 on Bioko Island

BackgroundPlasmodium falciparum apical membrane antigen-1 (PfAMA-1) is a promising candidate antigen for a blood-stage malaria vaccine. However, antigenic variation and diversity of PfAMA-1 are still major problems to design a universal malaria vaccine based on this antigen, especially against domain I (DI). Detail understanding of the PfAMA-1 gene polymorphism can provide useful information on this potential vaccine component. Here, general characteristics of genetic structure and the effect of natural selection of DIs among Bioko P. falciparum isolates were analysed.Methods214 blood samples were collected from Bioko Island patients with P. falciparum malaria between 2011 and 2017. A fragment spanning DI of PfAMA-1 was amplified by nested polymerase chain reaction and sequenced. Polymorphic characteristics and the effect of natural selection were analysed using MEGA 5.0, DnaSP 6.0 and Popart programs. Genetic diversity in 576 global PfAMA-1 DIs were also analysed. Protein function prediction of new amino acid mutation sites was performed using PolyPhen-2 program.Results131 different haplotypes of PfAMA-1 were identified in 214 Bioko Island P. falciparum isolates. Most amino acid changes identified on Bioko Island were found in C1L. 32 amino acid changes identified in PfAMA-1 sequences from Bioko Island were found in predicted RBC-binding sites, B cell epitopes or IUR regions. Overall patterns of amino acid changes of Bioko PfAMA-1 DIs were similar to those in global PfAMA-1 isolates. Differential amino acid substitution frequencies were observed for samples from different geographical regions. Eight new amino acid changes of Bioko island isolates were also identified and their three-dimensional protein structural consequences were predicted. Evidence for natural selection and recombination event were observed in global isolates.ConclusionsPatterns of nucleotide diversity and amino acid polymorphisms of Bioko Island isolates were similar to those of global PfAMA-1 DIs. Balancing natural selection across DIs might play a major role in generating genetic diversity in global isolates. Most amino acid changes in DIs occurred in predicted B-cell epitopes. Novel sites mapped on a three dimensional structure of PfAMA-1 showed that these regions were located at the corner. These results may provide significant value in the design of a malaria vaccine based on this antigen.

HIV infection drives IgM and IgG3 subclass bias in Plasmodium falciparum-specific and total immunoglobulin concentration in Western Kenya

BackgroundHIV infection is associated with more frequent and severe episodes of malaria and may be the result of altered malaria-specific B cell responses. However, it is poorly understood how HIV and the associated lymphopenia and immune activation affect malaria-specific antibody responses.MethodsHIV infected and uninfected adults were recruited from Bondo subcounty hospital in Western Kenya at the time of HIV testing (antiretroviral and co-trimoxazole prophylaxis naïve). Total and Plasmodium falciparum apical membrane antigen-1 (AMA1) and glutamate rich protein-R0 (GLURP-R0) specific IgM, IgG and IgG subclass concentrations was measured in 129 and 52 of recruited HIV-infected and uninfected individuals, respectively. In addition, HIV-1 viral load (VL), CD4+ T cell count, and C-reactive protein (CRP) concentration was quantified in study participants. Antibody levels were compared based on HIV status and the associations of antibody concentration with HIV-1 VL, CD4+ count, and CRP levels was measured using Spearman correlation testing.ResultsAmong study participants, concentrations of IgM, IgG1 and IgG3 antibodies to AMA1 and GLURP-R0 were higher in HIV infected individuals compared to uninfected individuals (all p < 0.001). The IgG3 to IgG1 ratio to both AMA1 and GLURP-R0 was also significantly higher in HIV-infected individuals (p = 0.02). In HIV-infected participants, HIV-1 VL and CRP were weakly correlated with AMA1 and GLURP-R0 specific IgM and IgG1 concentrations and total (not antigen specific) IgM, IgG, IgG1, and IgG3 concentrations (all p < 0.05), suggesting that these changes are related in part to viral load and inflammation.ConclusionsOverall, HIV infection leads to a total and malaria antigen-specific immunoglobulin production bias towards higher levels of IgM, IgG1, and IgG3, and HIV-1 viraemia and systemic inflammation are weakly correlated with these changes. Further assessments of antibody affinity and function and correlation with risk of clinical malaria, will help to better define the effects of HIV infection on clinical and biological immunity to malaria.

Sero-identification of the aetiologies of human malaria exposure (Plasmodium spp.) in the Limu Kossa District of Jimma Zone, South western Ethiopia

BackgroundMalaria remains a very important public health problem in Ethiopia. Currently, only Plasmodium falciparum and Plasmodium vivax are considered in the malaria diagnostic and treatment policies. However, the existence and prevalence of Plasmodium ovale spp. and Plasmodium malariae in Ethiopia have not been extensively investigated. The objective of this study was to use a multiplex IgG antibody detection assay to evaluate evidence for exposure to any of these four human malaria parasites among asymptomatic individuals.MethodsDried blood spots (DBS) were collected from 180 healthy study participants during a 2016 onchocerciasis survey in the Jimma Zone, southwest Ethiopia. IgG antibody reactivity was detected using a multiplex bead assay for seven Plasmodium antigens: P. falciparum circumsporozoite protein (CSP), P. falciparum apical membrane antigen-1 (AMA1), P. falciparum liver stage antigen-1 (LSA1), and homologs of the merozoite surface protein-1 (MSP1)-19kD antigens that are specific for P. falciparum, P. vivax, P. ovale spp. and P. malariae.ResultsOne hundred six participants (59%) were IgG seropositive for at least one of the Plasmodium antigens tested. The most frequent responses were against P. falciparum AMA1 (59, 33%) and P. vivax (55, 28%). However, IgG antibodies against P. ovale spp. and P. malariae were detected in 19 (11%) and 13 (7%) of the participants, respectively, providing serological evidence that P. malariae and P. ovale spp., which are rarely reported, may also be endemic in Jimma.ConclusionThe findings highlight the informative value of multiplex serology and the need to confirm whether P. malariae and P. ovale spp. are aetiologies of malaria in Ethiopia, which is critical for proper diagnosis and treatment.

Identification of the Binding Site of Apical Membrane Antigen 1 (AMA1) Inhibitors Using a Paramagnetic Probe.

Apical membrane antigen 1 (AMA1) is essential for the invasion of host cells by malaria parasites. Several small-molecule ligands have been shown to bind to a conserved hydrophobic cleft in Plasmodium falciparum AMA1. However, a lack of detailed structural information on the binding pose of these molecules has hindered their further optimisation as inhibitors. We have developed a spin-labelled peptide based on RON2, the native binding partner of AMA1, to probe the binding sites of compounds on PfAMA1. The crystal structure of this peptide bound to PfAMA1 shows that it binds at one end of the hydrophobic groove, leaving much of the binding site unoccupied and allowing fragment hits to bind without interference. In paramagnetic relaxation enhancement (PRE)-based NMR screening, the 1 H relaxation rates of compounds binding close to the probe were enhanced. Compounds experienced different degrees of PRE as a result of their different orientations relative to the spin label while bound to AMA1. Thus, PRE-derived distance constraints can be used to identify binding sites and guide further hit optimisation.

Population genetic structure of domain I of apical membrane antigen-1\xa0in Plasmodium falciparum\xa0isolates from Hazara division of Pakistan

BackgroundThe Plasmodium falciparum apical membrane antigen-1 (PfAMA1) is considered as an ideal vaccine candidate for malaria control due to its high level of immunogenicity and essential role in parasite survival. Among the three domains of PfAMA1 protein, hyper-variable region (HVR) of domain I is the most immunogenic. The present study was conducted to evaluate the extent of genetic diversity across HVR domain I of the pfama1 gene in P. falciparum isolates from Hazara division of Pakistan.MethodsThe HVR domain I of the pfama1 was amplified and sequenced from 20 P. falciparum positive cases from Hazara division of Pakistan. The sequences were analysed in context of global population data of P. falciparum from nine malaria endemic countries. The DNA sequence reads quality assessment, reads assembling, sequences alignment/phylogenetic and population genetic analyses were performed using Staden, Lasergene v. 7.1, MEGA7 and DnaSP v.5 software packages respectively.ResultsTotal 14 mutations were found in Pakistani isolates with 12 parsimony informative sites. During comparison with global isolates, a novel non-synonymous mutation (Y240F) was found specifically in a single Pakistani sample with 5% frequency. The less number of mutations, haplotypes, recombination and low pairwise nucleotide differences revealed tightly linked uniform genetic structure with low genetic diversity at HVR domain I of pfama1 among P. falciparum isolates from Hazara region of Pakistan. This uniform genetic structure may be shaped across Pakistani P. falciparum isolates by bottleneck or natural selection events.ConclusionThe Pakistani P. falciparum isolates were found to maintain a distinct genetic pattern at HVR pfama1 with some extent of genetic relationship with geographically close Myanmar and Indian samples. However, the exact pattern of gene flow and demographic events may infer from whole genome sequence data with large sample size of P. falciparum collected from broad area of Pakistan.

Antibody responses to P. falciparum Apical Membrane Antigen 1(AMA-1) in relation to haemoglobin S (HbS), HbC, G6PD and ABO blood groups among Fulani and Masaleit living in Western Sudan

Fulani and Masaleit are two sympatric ethnic groups in western Sudan who are characterised by marked differences in susceptibility to Plasmodium falciparum malaria. It has been demonstrated that Glucose-6-phosphate dehydrogenase (G6PD) deficiency and Sickle cell trait HbAS carriers are protected from the most severe forms of malaria. This study aimed to investigate a set of specific IgG subclasses against P. falciparum Apical Membrane Antigen 1 (AMA-1 3D7), haemoglobin variants and (G6PD) in association with malaria susceptibility among Fulani ethnic group compared to sympatric ethnic group living in Western Sudan. A total of 124 children aged 5–9 years from each tribe living in an area of hyper-endemic P. falciparum unstable malaria transmission were recruited and genotyped for the haemoglobin (Hb) genes, (G6PD) and (ABO) blood groups. Furthermore, the level of plasma IgG antibody subclasses against P. falciparum antigen (AMA-1) were measured using enzyme linked immunosorbent assays (ELISA). Higher levels of anti-malarial IgG1, IgG2 and IgG3 but not IgG4 antibody were found in Fulani when compared to Masaleit. Individuals carrying the HbCC phenotype were significantly associated with higher levels of IgG1 and IgG2. Furthermore, individuals having the HbAS phenotype were associated with higher levels of specific IgG2 and IgG4 antibodies. In addition, patients with G6PD A/A genotype were associated with higher levels of specific IgG2 antibody compared with those carrying the A/G and G/G genotypes. The results indicate that the Fulani ethnic group show lower frequency of HbAS, HbSS and HbAC compared to the Masaleit ethnic group. The inter-ethnic analysis shows no statistically significant difference in G6PD genotypes (P value = 0.791). However, the intra-ethnic analysis indicates that both ethnic groups have less A/A genotypes and (A) allele frequency of G6PD compared to G/G genotypes, while the HbSA genotype was associated with higher levels of IgG2 (AMA-1) and IgG4 antibodies. In addition, patients carrying the G6PD A/A genotype were associated with higher levels of specific IgG2 antibody compared with those carrying the A/G and G/G genotypes. The present results revealed that the Fulani ethnic group has statistically significantly lower frequency of abnormal haemoglobin resistant to malaria infection compared to the Masaleit ethnic group.

Population genetic structure and natural selection of Plasmodium falciparum apical membrane antigen-1 in Myanmar isolates

BackgroundPlasmodium falciparum apical membrane antigen-1 (PfAMA-1) is one of leading blood stage malaria vaccine candidates. However, genetic variation and antigenic diversity identified in global PfAMA-1 are major hurdles in the development of an effective vaccine based on this antigen. In this study, genetic structure and the effect of natural selection of PfAMA-1 among Myanmar P. falciparum isolates were analysed.MethodsBlood samples were collected from 58 Myanmar patients with falciparum malaria. Full-length PfAMA-1 gene was amplified by polymerase chain reaction and cloned into a TA cloning vector. PfAMA-1 sequence of each isolate was sequenced. Polymorphic characteristics and effect of natural selection were analysed with using DNASTAR, MEGA4, and DnaSP programs. Polymorphic nature and natural selection in 459 global PfAMA-1 were also analysed.ResultsThirty-seven different haplotypes of PfAMA-1 were identified in 58 Myanmar P. falciparum isolates. Most amino acid changes identified in Myanmar PfAMA-1 were found in domains I and III. Overall patterns of amino acid changes in Myanmar PfAMA-1 were similar to those in global PfAMA-1. However, frequencies of amino acid changes differed by country. Novel amino acid changes in Myanmar PfAMA-1 were also identified. Evidences for natural selection and recombination event were observed in global PfAMA-1. Among 51 commonly identified amino acid changes in global PfAMA-1 sequences, 43 were found in predicted RBC-binding sites, B-cell epitopes, or IUR regions.ConclusionsMyanmar PfAMA-1 showed similar patterns of nucleotide diversity and amino acid polymorphisms compared to those of global PfAMA-1. Balancing natural selection and intragenic recombination across PfAMA-1 are likely to play major roles in generating genetic diversity in global PfAMA-1. Most common amino acid changes in global PfAMA-1 were located in predicted B-cell epitopes where high levels of nucleotide diversity and balancing natural selection were found. These results highlight the strong selective pressure of host immunity on the PfAMA-1 gene. These results have significant implications in understanding the nature of Myanmar PfAMA-1 along with global PfAMA-1. They also provide useful information for the development of effective malaria vaccine based on this antigen.

Variation in the immune responses against Plasmodium falciparum merozoite surface protein-1 and apical membrane antigen-1 in children residing in the different epidemiological strata of malaria in Cameroon

BackgroundStudies to assess the immune responses against malaria in Cameroonian children are limited. The purpose of this study was to assess the immune responses against Plasmodium falciparum merozoite surface protein-1 (MSP-119) and apical membrane antigen-1 (AMA-1) in children residing in the different epidemiological strata of malaria in Cameroon.MethodsIn a cross-sectional survey performed between April and July 2015, 602 children between 2 and 15 years (mean ± SD = 5.7 ± 3.7), comprising 319 (53%) males were enrolled from five epidemiological strata of malaria in Cameroon including: the sudano-sahelian (SS) strata, the high inland plateau (HIP) strata, the south Cameroonian equatorial forest (SCEF) strata, the high western plateau (HWP) strata, and the coastal (C) strata. The children were screened for clinical malaria (defined by malaria parasitaemia ≥ 5000 parasites/µl plus axillary temperature ≥ 37.5 °C). Their antibody responses were measured against P. falciparum MSP-119 and AMA-1 vaccine candidate antigens using standard ELISA technique.ResultsA majority of the participants were IgG responders 72.1% (95% CI 68.3–75.6). The proportion of responders was higher in females (p = 0.002) and in children aged 10 years and above (p = 0.005). The proportion of responders was highest in Limbe (C strata) and lowest in Ngaoundere (HIP strata) (p < 0.0001). Similarly, the mean IgG antibody levels were higher in children aged 10 years and above (p < 0.0001) and in Limbe (p = 0.001). The IgG antibody levels against AMA-1 were higher in females (p = 0.028), meanwhile no gender disparity was observed with MSP-1. Furthermore the risk of clinical malaria (p < 0.0001) and the mean parasite density (p = 0.035) were higher in IgG non-responders.ConclusionA high proportion of IgG responders was observed in this study, suggesting a high degree exposure of the target population to malaria parasites. The immune responses varied considerably across the different strata: the highest levels observed in the C strata and the lowest in the HIP strata. Furthermore, malaria transmission in Cameroon could be categorized into two major groups based on the serological reaction of the children: the southern (comprising C and SCEF strata) and northern (comprising HWP, HIP and SS strata) groups. These findings may have significant implications in the design of future trials for evaluating malaria vaccine candidates in Cameroon.

Cross-reactivity between apical membrane antgen 1 and rhoptry neck protein 2 in P. vivax and P. falciparum: A structural and binding study.

Malaria, a disease endemic in many tropical and subtropical regions, is caused by infection of the erythrocyte by the apicomplexan parasite Plasmodium. Host-cell invasion is a complex process but two Plasmodium proteins, Apical Membrane Antigen 1 (AMA1) and the Rhoptry Neck protein complex (RON), play a key role. AMA1, present on the surface of the parasite, binds tightly to the RON2 component of the RON protein complex, which is inserted into the erythrocyte membrane during invasion. Blocking the AMA1-RON2 interaction with antibodies or peptides inhibits invasion, underlining its importance in the Plasmodium life cycle and as a target for therapeutic strategies. We describe the crystal structure of the complex formed between AMA1 from P. vivax (PvAMA1) and a peptide derived from the externally exposed region of P. vivax RON2 (PvRON2sp1), and of the heterocomplex formed between P. falciparum AMA1 (PfAMA1) and PvRON2sp1. Binding studies show that the affinity of PvRON2sp1 for PvAMA1 is weaker than that previously reported for the PfRON2sp1-PfAMA1 association. Moreover, while PvRON2sp1 shows strong cross-reactivity with PfAMA1, PfRON2sp1 displays no detectable interaction with PvAMA1. The structures show that the equivalent residues PvRON2-Thr2055 and PfRON2-Arg2041 largely account for this pattern of reactivity.

The establishment of a WHO Reference Reagent for anti-malaria (Plasmodium falciparum) human serum

BackgroundAt a World Health Organization (WHO) sponsored meeting it was concluded that there is an urgent need for a reference preparation that contains antibodies against malaria antigens in order to support serology studies and vaccine development. It was proposed that this reference would take the form of a lyophilized serum or plasma pool from a malaria-endemic area. In response, an immunoassay standard, comprising defibrinated human plasma has been prepared and evaluated in a collaborative study.ResultsA pool of human plasma from a malaria endemic region was collected from 140 single plasma donations selected for reactivity to Plasmodium falciparum apical membrane antigen-1 (AMA-1) and merozoite surface proteins (MSP-119, MSP-142, MSP-2 and MSP-3). This pool was defibrinated, filled and freeze dried into a single batch of ampoules to yield a stable source of naturally occurring antibodies to P. falciparum. The preparation was evaluated by an enzyme-linked immunosorbent assay (ELISA) in a collaborative study with sixteen participants from twelve different countries. This anti-malaria human serum preparation (NIBSC Code: 10/198) was adopted by the WHO Expert Committee on Biological Standardization (ECBS) in October 2014, as the first WHO reference reagent for anti-malaria (Plasmodium falciparum) human serum with an assigned arbitrary unitage of 100 units (U) per ampoule.ConclusionAnalysis of the reference reagent in a collaborative study has demonstrated the benefit of this preparation for the reduction in inter- and intra-laboratory variability in ELISA. Whilst locally sourced pools are regularly use for harmonization both within and between a few laboratories, the presence of a WHO-endorsed reference reagent should enable optimal harmonization of malaria serological assays either by direct use of the reference reagent or calibration of local standards against this WHO reference. The intended uses of this reference reagent, a multivalent preparation, are (1) to allow cross-comparisons of results of vaccine trials performed in different centres/with different products; (2) to facilitate standardization and harmonization of immunological assays used in epidemiology research; and (3) to allow optimization and validation of immunological assays used in malaria vaccine development.

THE EFFECT OF HELMINTH CO-INFECTION ON MALARIA-SPECIFIC IMMUNOGLOBULIN G RESPONSES

BackgroundMalaria and helminthiases overlap extensively in their epidemiological distributions, and co-infections are common. Helminth infection has a profound effect on the immune system such as the induction of immuno-regulatory mechanisms...

Serology reveals heterogeneity of Plasmodium falciparum transmission in northeastern South Africa: implications for malaria elimination

BackgroundIt is widely acknowledged that modifications to existing control interventions are required if South Africa is to achieve malaria elimination. Targeting indoor residual spraying (IRS) to areas where cases have been detected is one strategy currently under investigation in northeastern South Africa. This seroprevalence baseline study, nested within a targeted IRS trial, was undertaken to provide insights into malaria transmission dynamics in South Africa and evaluate whether sero-epidemiological practices have the potential to be routinely incorporated into elimination programmes.MethodsFilter-paper blood spots, demographic and household survey data were collected from 2710 randomly selected households in 56 study wards located in the municipalities of Ba-Phalaborwa and Bushbuckridge. Blood spots were assayed for Plasmodium falciparum apical membrane antigen-1 and merozoite surface protein-119 blood-stage antigens using an enzyme linked immunosorbent assay. Seroprevalence data were analysed using a reverse catalytic model to determine malaria seroconversion rates (SCR). Geospatial cluster analysis was used to investigate transmission heterogeneity while random effects logistic regression identified risk factors associated with malaria exposure.ResultsThe overall SCR across the entire study site was 0.012 (95% CI 0.008–0.017) per year. Contrasting SCRs, corresponding to distinct geographical regions across the study site, ranging from <0.001 (95% CI <0.001–0.005) to 0.022 (95% CI 0.008–0.062) per annum revealed prominent transmission heterogeneity. Geospatial cluster analysis of household seroprevalence and age-adjusted antibody responses detected statistically significant (p < 0.05) spatial clusters of P. falciparum exposure. Formal secondary education was associated with lower malaria exposure in the sampled population (AOR 0.72, 95% CI 0.56–0.95, p = 0.018).ConclusionsAlthough overall transmission intensity and exposure to malaria was low across both study sites, malaria transmission intensity was highly heterogeneous and associated with low socio-economic status in the region. Findings suggest focal targeting of interventions has the potential to be an appropriate strategy to deploy in South Africa. Furthermore, routinely incorporating sero-epidemiological practices into elimination programmes may prove useful in monitoring malaria transmission intensity in South Africa, and other countries striving for malaria elimination.

Structure–Activity Studies of β-Hairpin Peptide Inhibitors of the Plasmodium falciparum AMA1–RON2 Interaction

The interaction between apical membrane antigen 1 (AMA1) and rhoptry neck protein 2 (RON2) plays a key role in the invasion of red blood cells by Plasmodium parasites. Disruption of this critical protein–protein interaction represents a promising avenue for antimalarial drug discovery. In this work, we exploited a 13-residue β-hairpin based on the C-terminal loop of RON2 to probe a conserved binding site on Plasmodium falciparum AMA1. A series of mutations was synthetically engineered into β-hairpin peptides to establish structure–activity relationships. The best mutations improved the binding affinity of the β-hairpin peptide by ~7-fold for 3D7 AMA1 and ~14-fold for FVO AMA1. We determined the crystal structures of several β-hairpin peptides in complex with AMA1 in order to define the structural features and specific interactions that contribute to improved binding affinity. The same mutations in the longer RON2sp2 peptide (residues 2027–2055 of RON2) increased the binding affinity by >30-fold for 3D7 and FVO AMA1, producing KD values of 2.1nM and 0.4nM, respectively. To our knowledge, this is the most potent strain-transcending peptide reported to date and represents a valuable tool to characterize the AMA1–RON2 interaction.

Measurement of ex vivo ELISpot interferon-gamma recall responses to Plasmodium falciparum AMA1 and CSP in Ghanaian adults with natural exposure to malaria.

BackgroundMalaria eradication requires a concerted approach involving all available control tools, and an effective vaccine would complement these efforts. An effective malaria vaccine should be able to induce protective immune responses in a genetically diverse population. Identification of immunodominant T cell epitopes will assist in determining if candidate vaccines will be immunogenic in malaria-endemic areas. This study therefore investigated whether class I-restricted T cell epitopes of two leading malaria vaccine antigens, Plasmodium falciparum circumsporozoite protein (CSP) and apical membrane antigen-1 (AMA1), could recall T cell interferon-γ responses from naturally exposed subjects using ex vivo ELISpot assays.MethodsThirty-five subjects aged between 24 and 43 years were recruited from a malaria-endemic urban community of Ghana in 2011, and their peripheral blood mononuclear cells (PBMCs) were tested in ELISpot IFN-γ assays against overlapping 15mer peptide pools spanning the entire CSP and AMA1 antigens, and 9–10mer peptide epitope mixtures that included previously identified and/or predicted human leukocyte antigen (HLA) class 1-restricted epitopes from same two antigens.ResultsFor CSP, 26 % of subjects responded to at least one of the nine 15mer peptide pools whilst 17 % responded to at least one of the five 9–10mer HLA-restricted epitope mixtures. For AMA1, 63 % of subjects responded to at least one of the 12 AMA1 15mer peptide pools and 51 % responded to at least one of the six 9–10mer HLA-restricted epitope mixtures. Following analysis of data from the two sets of peptide pools, along with bioinformatics predictions of class I-restricted epitopes and the HLA supertypes expressed by a subset of study subjects, peptide pools that may contain epitopes recognized by multiple HLA supertypes were identified. Collectively, these results suggest that natural transmission elicits ELISpot IFN-γ activities to class 1-restricted epitopes that are largely HLA-promiscuous.ConclusionsThese results generally demonstrate that CSP and AMA1 peptides recalled ELISpot IFN-γ responses from naturally exposed individuals and that both CSP and AMA1 contain diverse class 1-restricted epitopes that are HLA-promiscuous and are widely recognized in this population.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-016-1098-8) contains supplementary material, which is available to authorized users.

Potential Impact of Seasonal Malaria Chemoprevention on the Acquisition of Antibodies Against Glutamate-Rich Protein and Apical Membrane Antigen 1 in Children Living in Southern Senegal.

Seasonal malaria chemoprevention (SMC) is defined as the intermittent administration of full treatment courses of an antimalarial drug to children during the peak of malaria transmission season with the aim of preventing malaria-associated mortality and morbidity. SMC using sulfadoxine–pyrimethamine (SP) combined with amodiaquine (AQ) is a promising strategy to control malaria morbidity in areas of highly seasonal malaria transmission. However, a concern is whether SMC can delay the natural acquisition of immunity toward malaria parasites in areas with intense SMC delivery. To investigate this, total IgG antibody (Ab) responses to Plasmodium falciparum antigens glutamate-rich protein R0 (GLURP-R0) and apical membrane antigen 1 (AMA-1) were measured by enzyme-linked immunosorbent assay in Senegalese children under the age of 10 years in 2010 living in Saraya and Velingara districts (with SMC using SP+AQ [SMC+] since 2007) and Tambacounda district (without SMC (SMC−)). For both P. falciparum antigens, total IgG response were significantly higher in the SMC− compared with the SMC+ group (for GLURP-R0, P < 0.001 and for AMA-1, P = 0.001). There was as well a nonsignificant tendency for higher percentage of positive responders in the SMC− compared with the SMC+ group (for GLURP-R0: 22.2% versus 14.4%, respectively [P = 0.06]; for AMA-1: 45.6% versus 40.0%, respectively [P = 0.24]). Results suggest that long-term malaria chemoprevention by SMC/SP+AQ have limited impact on the development of acquired immunity, as tested using the P. falciparum antigens GLURP-R0 and AMA-1. However, other factors, not measured in this study, may interfere as well.