Atypical memory B cell frequency correlates with antibody breadth and function in malaria immune adults.

Clinical immunity to malaria develops slowly after repeated episodes of infection and antibodies are essential in naturally acquired immunity against malaria. However, chronic exposure to malaria has been linked to perturbation in B-cell homeostasis with the accumulation of atypical memory B cells. It is unclear how perturbations in B cell subsets influence antibody breadth, avidity, and function in individuals naturally exposed to malaria. We show that individuals living in high malaria transmission regions in Ghana have higher Plasmodium falciparum merozoite antigen-specific antibodies and an increased antibody breadth score but lower antibody avidities relative to low transmission regions. The frequency of circulating atypical memory B cells is positively associated with an individual's antibody breadth. In vitro growth inhibition is independent of the ability to bind to free merozoites but associated with the breadth of antibody reactivity in an individual. Taken together, our data shows that repeated malaria episodes hamper the development of high avid antibodies which is compensated for by an increase in antibody breadth. Our results provide evidence to reinforce the idea that in regions with high malaria prevalence, repeated malaria infections lead to the broadening of antibody diversity and the continued presence of atypical memory B cell populations.

Clinical and laboratory characteristics of children with sickle cell disease on hydroxyurea treated with artemether-lumefantrine for acute uncomplicated malaria.

Introduction: Limited information exists on any interactions between hydroxyurea (HU) and antimalarials in sickle cell disease (SCD). We evaluated changes in clinical and laboratory parameters among children with SCD on HU therapy treated with artemether-lumefantrine (AL) for acute uncomplicated malaria (UM). Methods: A prospective, non-randomized, pilot study of 127 children with SCD (23, UM; 104, steady state) were recruited from three hospitals in Accra. UM participants were treated with standard doses of AL and followed up, on days 1, 2, 3, 7, 14, and 28. Venous blood was collected at baseline and follow-up days in participants with UM for determination of malaria parasitaemia, full blood count, reticulocytes, and clinical chemistry. Further, Plasmodium falciparum identification of rapid diagnostic test (RDT) positive samples was done using nested polymerase chain reaction (PCR). Results: Among SCD participants with UM, admission temperature, neutrophils, alanine-aminotransferase, gamma-glutamyl-transferase, and haemoglobin significantly differed between HU recipients (HU+) and steady state, while white blood cell, neutrophils, reticulocytes, bilirubin, urea, and temperature differed significantly between non-HU recipients (no-HU), and steady state. Mean parasitaemia (HU+, 2930.3 vs. no-HU, 1,060, p = 0.74) and adverse events (HU+, 13.9% vs. no-HU, 14.3%), were comparable (p = 0.94). Day 28 reticulocyte count was higher in the HU+ (0.24) (0.17 to 0.37) vs. no-HU, [0.15 (0.09 to 0.27), p = 0.022]. Significant differences in lymphocyte [HU+ 2.74 95% CI (-5.38 to 58.57) vs. no-HU -0.34 (-3.19 to 4.44), p = 0.024]; bilirubin [HU+, -4.44 (-16.36 to 20.74) vs. no-HU -18.37 (-108.79 to -7.16)]; and alanine aminotransferase, [HU+, -4.00 (-48.55 to 6.00) vs. no-HU, 7.00 (-22.00 to 22.00)] were observed during follow up. Conclusion: Parasite clearance and adverse event occurrence were comparable between SCD children treated with AL irrespective of HU status. However, distinct patterns of changes in laboratory indices suggest the need for larger, more focused studies.

Inflammatory cytokines as potential biomarkers for early diagnosis of severe malaria in children in Ghana.

BACKGROUND: Severe malaria (SM) is a fatal multi-system disease which accounted for an estimated 619,000 deaths in 2021. Less than 30% of children presenting with SM are diagnosed and treated promptly, resulting in increased mortality and neurologic impairments in survivors. Studies have identified cytokine profiles that differentiate the various clinical manifestations of malaria (severe and uncomplicated). However, the diagnostic capability of these cytokines in differentiating between the disease states in terms of cut-off values has not yet been determined. METHODS: The plasma levels of 22 pro-inflammatory cytokines (Eotaxin/CCL 11, interferon-gamma (IFN-γ), interleukin (IL)- 2, IL-6, IL-1ß, IL-12p40/p70, IL-17A, RANTES, MCP-1, IL-15, IL-5, IL-1RA, IL-2R, IFN-α, IP-10, TNF, MIG, MIP-1α, MIP-1ß, IL-7, IL-8 and Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF), and 3 anti-inflammatory cytokines-(IL-4, IL-13 and IL-10) in patients with SM, uncomplicated malaria (UM) and other febrile conditions, were measured and compared using the Human Cytokine Magnetic 25-Plex Panel. The receiver operating characteristic (ROC) curve analysis was used to determine the diagnostic value of these cytokines. RESULTS: The level of the pro-inflammatory cytokine, IL-17A, was significantly higher in the SM group as compared to the UM group. Levels of the anti-inflammatory cytokines however did not differ significantly among the SM and UM groups. Only IL-1ß and IL-17A showed good diagnostic potential after ROC curve analysis. CONCLUSION: The data show that levels of pro-inflammatory cytokines correlate with malaria disease severity. IL-1ß and IL-17A showed good diagnostic potentials and can be considered for use in clinical practice to target treatment.

Cryo-EM reveals the conformational epitope of human monoclonal antibody PAM1.4 broadly reacting with polymorphic malarial protein VAR2CSA.

Malaria during pregnancy is a major global health problem caused by infection with Plasmodium falciparum parasites. Severe effects arise from the accumulation of infected erythrocytes in the placenta. Here, erythrocytes infected by late blood-stage parasites adhere to placental chondroitin sulphate A (CS) via VAR2CSA-type P. falciparum erythrocyte membrane protein 1 (PfEMP1) adhesion proteins. Immunity to placental malaria is acquired through exposure and mediated through antibodies to VAR2CSA. Through evolution, the VAR2CSA proteins have diversified in sequence to escape immune recognition but retained their overall macromolecular structure to maintain CS binding affinity. This structural conservation may also have allowed development of broadly reactive antibodies to VAR2CSA in immune women. Here we show the negative stain and cryo-EM structure of the only known broadly reactive human monoclonal antibody, PAM1.4, in complex with VAR2CSA. The data shows how PAM1.4's broad VAR2CSA reactivity is achieved through interactions with multiple conserved residues of different sub-domains forming conformational epitope distant from the CS binding site on the VAR2CSA core structure. Thus, while PAM1.4 may represent a class of antibodies mediating placental malaria immunity by inducing phagocytosis or NK cell-mediated cytotoxicity, it is likely that broadly CS binding-inhibitory antibodies target other epitopes at the CS binding site. Insights on both types of broadly reactive monoclonal antibodies may aid the development of a vaccine against placental malaria.

Perturbations in the T cell receptor ß repertoire during malaria infection in children: A preliminary study.

The changes occurring in the T cell repertoire during clinical malaria infection in children remain unknown. In this study, we undertook the first detailed comparative study of the T cell repertoire in African children with and without clinical malaria to test the hypothesis that clonotypic expansions that occur during P. falciparum infection will contribute to the generation of a T cell repertoire that is unique to each disease state. We profiled the complementarity-determining region 3 (CDR3) of the TCRß chain sequences from children with Plasmodium falciparum infections (asymptomatic, uncomplicated and severe malaria) and compared these with sequences from healthy children. Interestingly, we discovered that children with symptomatic malaria have a lower TCR diversity and frequency of shared (or "public") TCR sequences compared to asymptomatic children. Also, TCR diversity was inversely associated with parasitemia. Furthermore, by clustering TCR sequences based on their predicted antigen specificities, we identified a specificity cluster, with a 4-mer amino acid motif, that is overrepresented in the asymptomatic group compared to the diseased groups. Further investigations into this finding may help in delineating important antigenic targets for vaccine and therapeutic development. The results show that the T cell repertoire in children is altered during malaria, suggesting that exposure to P. falciparum antigens disrupts the adaptive immune response, which is an underlying feature of the disease.

Identification and Enumeration of Plasmodium falciparum Parasites by Light Microscopy.

Estimating malaria parasite density is important for patient care and management in rural and urban health centers in endemic areas. Here, we describe methodologically the protocols and methods to identify and enumerate Plasmodium falciparum parasites from infected blood using light microscopy. We provide step-by-step protocols and evaluate any possible drawbacks that may limit the methods and prospects of using light microscopy in diagnosing malaria.

Collection and Cryopreservation of Plasmodium falciparum Clinical Isolates in the Field.

P. falciparum causes the most severe form of malaria in younger children and pregnant women. In vitro culture systems allow researchers to understand parasite biology, elucidate mechanism of host immunity and test efficacy of antimalarial agents or vaccines in preclinical studies. Most laboratory-adapted parasite strains predate the emergence of artemisinin-based drug combinations and mainly originate from Asia or Europe. To fully understand the biochemical and phenotypic characteristics of parasites, it is imperative that researchers are able to culture parasites circulating in an area to unravel any geographical differences at the population level. Ex vivo culturing of clinical isolates can be challenging when collecting samples in the field and requires technical expertise and equipment. To overcome this challenge, clinical isolates are cryopreserved in the field and transported to a laboratory for in vitro studies. In this protocol, we describe different methods of cryopreserving P. falciparum isolates in the field and thawing them for subsequent in vitro culture.

Analysis of allelic cross-reactivity of monoclonal IgG antibodies by a multiplexed reverse FluoroSpot assay.

The issue of antibody cross-reactivity is of central importance in immunology, and not least in protective immunity to Plasmodium falciparum malaria, where key antigens show substantial allelic variation (polymorphism). However, serological analysis often does not allow the distinction between true cross-reactivity (one antibody recognizing multiple antigen variants) and apparent cross-reactivity (presence of multiple variant-specific antibodies), as it requires analysis at the single B-cell/monoclonal antibody level. ELISpot is an assay that enables that, and a recently developed multiplexed variant of ELISpot (FluoroSpot) facilitates simultaneous assessment of B-cell/antibody reactivity to several different antigens. In this study, we present a further enhancement of this assay that makes direct analysis of monoclonal antibody-level cross-reactivity with allelic variants feasible. Using VAR2CSA-type PfEMP1-a notoriously polymorphic antigen involved in the pathogenesis of placental malaria-as a model, we demonstrate the robustness of the assay and its applicability to analysis of true cross-reactivity of monoclonal VAR2CSA-specific antibodies in naturally exposed individuals. The assay is adaptable to the analysis of other polymorphic antigens, rendering it a powerful tool in studies of immunity to malaria and many other diseases.

Cytokines as Potential Biomarkers for Differential Diagnosis of Sepsis and Other Non-Septic Disease Conditions.

Sepsis defined as a dysregulated immune response is a major cause of morbidity in children. In sub-Saharan Africa, the clinical features of sepsis overlap with other frequent infections such as malaria, thus sepsis is usually misdiagnosed in the absence of confirmatory tests. Therefore, it becomes necessary to identify biomarkers that can be used to distinguish sepsis from other infectious diseases. We measured and compared the plasma levels of 18 cytokines (Th1 [GM-CSF, IFN-γ, TNF-α, IL-1ß, 1L-2, IL-6, IL-8, IL-12/IL-23p40, IL-15], Th2[IL-4, IL-5, IL-13), Th17 [IL17A], Regulatory cytokine (IL-10) and 7 chemokines (MCP-1/CCL2, MIP-1α/CCL3, MIP-1ß/CCL4, RANTES/CCL5, Eotaxin/CCL11, MIG/CXCL9 and IP-10/CXCL10 using the Human Cytokine Magnetic 25-Plex Panel in plasma samples obtained from children with sepsis, clinical malaria and other febrile conditions. Children with sepsis had significantly higher levels of IL-1ß, IL-12 and IL-17A compared to febrile controls but lower levels of MIP1-ß/CCL4, RANTES/CCL5 and IP10/CXCL10 when compared to children with malaria and febrile controls. Even though levels of most inflammatory responses were higher in malaria compared to sepsis, children with sepsis had a higher pro-inflammatory to anti-inflammatory ratio which seemed to be mediated by mostly monocytes. A principal component analysis and a receiver operator characteristic curve analysis, identified seven potential biomarkers; IL-1ß, IL-7, IL-12, IL-1RA, RANTES/CCL5, MIP1ß/CCL4 and IP10/CXCL10 that could discriminate children with sepsis from clinical malaria and other febrile conditions. The data suggests that sepsis is associated with a higher pro-inflammatory environment. These pro-inflammatory cytokines/chemokines could further be evaluated for their diagnostic potential to differentiate sepsis from malaria and other febrile conditions in areas burdened with infectious diseases.

High infectious disease burden as a basis for the observed high frequency of asymptomatic SARS-CoV-2 infections in sub-Saharan Africa.

Following the coronavirus outbreaks described as severe acute respiratory syndrome (SARS) in 2003 and the Middle East respiratory syndrome (MERS) in 2012, the world has again been challenged by yet another corona virus, named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). SARS-CoV-2 infections were first detected in a Chinese Province in December 2019 and then declared a pandemic by the World Health Organization in March 2020. An infection caused by SARS-CoV-2 may result in asymptomatic, uncomplicated or fatal coronavirus disease 2019 (COVID-19). Fatal disease has been linked with the uncontrolled "cytokine storm" manifesting with complications mostly in people with underlying cardiovascular and pulmonary disease conditions. The severity of COVID-19 disease and the associated mortality has been disproportionately lower in terms of number of cases and deaths in Africa and also Asia in comparison to Europe and North America. Also, persons of colour residing in Europe and North America have been identified as a highly susceptible population due to a combination of several socioeconomic factors and poor access to quality healthcare. Interestingly, this has not been the case in sub-Saharan Africa where majority of the population are even more deprived of the aforementioned factors. On the contrary, sub-Saharan Africa has recorded the lowest levels of mortality and morbidity associated with the disease, and an overwhelming proportion of infections are asymptomatic. Whilst it can be argued that these lower number of cases in Africa may be due to challenges associated with the diagnosis of the disease such as lack of trained personnel and infrastructure, the number of persons who get infected and develop symptoms is proportionally lower than those who are asymptomatic, including asymptomatic cases that are never diagnosed. This review discusses the most probable reasons for the significantly fewer cases of severe COVID-19 disease and deaths in sub-Saharan Africa.

Suitability of IgG responses to multiple Plasmodium falciparum antigens as markers of transmission intensity and pattern.

Detection of antibody reactivity to appropriate, specific parasite antigens may constitute a sensitive and cost-effective alternative to current tools to monitor malaria transmission across different endemicity settings. This study aimed to determine the suitability of IgG responses to a number of P. falciparum antigens as markers of transmission intensity and pattern. Antibody responses to multiple malaria antigens were determined in 905 participants aged 1-12 years from three districts with low (Keta), medium (Hohoe) and high (Krachi) transmission intensity in the Volta region of Ghana. Blood film microscopy slides and dry blood spots (DBS) were obtained for parasitaemia detection and antibody measurement, respectively. Sera were eluted from DBS and levels of IgG specific for 10 malaria antigens determined by a multiplex assay. Results were compared within and among the districts. Total IgG responses to MSPDBL1, MSPDBLLeucine, MSP2-FC27, RAMA, and PfRh2a and PfRh2b were higher in Krachi than in Hohoe and Keta. Seroprevalence of IgG specific for MSPDBLLeucine, RON4, and PfRh2b were also highest in Krachi. Responses to RALP-1, PfRh2a and PfRh2b were associated with patent but asymptomatic parasitaemia in Keta, while responses to MSPDBL1, MSPDBLLeucine, MSP2-FC27, RAMA, Rh2-2030, and PfRh2b were associated with parasite carriage in Hohoe, but not in Krachi. Using ROC analysis, only PfRh2b was found to predict patent, but asymptomatic, parasitaemia in Keta and Hohoe. Antibody breadth correlated positively with age (r = 0.29, p<0.0001) and parasitaemia (ß = 3.91; CI = 1.53 to 6.29), and medium to high transmission (p<0.0001). Our findings suggest differences in malaria-specific antibody responses across the three transmission zones and that PfRh2b has potential as a marker of malaria transmission intensity and pattern. This could have implications for malaria control programs and vaccine trials.

Acute Phase Responses Vary Between Children of HbAS and HbAA Genotypes During Plasmodium falciparum Infection.

PURPOSE: Haemoglobin genotype S is known to offer protection against Plasmodium falciparum infections but the mechanism underlying this protection is not completely understood. Associated changes in acute phase proteins (APPs) during Plasmodium falciparum infections between Haemoglobin AA (HbAA) and Haemoglobin AS (HbAS) individuals also remain unclear. This study aimed to evaluate changes in three APPs and full blood count (FBC) indices of HbAA and HbAS children during Plasmodium falciparum infection. METHODS: Venous blood was collected from three hundred and twenty children (6 months to 15 years) in Begoro in Fanteakwa District of Ghana during a cross-sectional study. Full blood count (FBC) indices were measured and levels of previously investigated APPs in malaria patients; C-reactive protein (CRP), ferritin and transferrin measured using Enzyme-Linked Immunosorbent Assays. RESULTS: Among the HbAA and HbAS children, levels of CRP and ferritin were higher in malaria positive children as compared to those who did not have malaria. The mean CRP levels were significantly higher among HbAA children (p=0.2e-08) as compared to the HbAS children (p=0.43). Levels of transferrin reduced in both HbAA and HbAS children with malaria, but the difference was only significant among HbAA children (p=0.0038), as compared to the HbAS children. No significant differences were observed in ferritin levels between HbAA and HbAS children in both malaria negative (p=0.76) and positive (p=0.26) children. Of the full blood count indices measured, red blood cell count (p=0.044) and haemoglobin (Hb) levels (p=0.017) differed between HbAA and HbAS in those without malaria, with higher RBC counts and lower Hb levels found in HbAS children. In contrast, during malaria, lymphocyte and platelet counts were elevated, whilst granulocytes and Mean Cell Haematocrit counts were reduced among children of the HbAS genotypes. CONCLUSION: Significant changes in APPs were found in HbAA children during malaria as compared to HbAS children, possibly due to differences in malaria-induced inflammation levels. This suggests that the HbAS genotype is associated with better control of P. falciparum infection-induced inflammatory response than HbAA genotype.

Ex vivo Sensitivity Profile of Plasmodium falciparum Clinical Isolates to a Panel of Antimalarial Drugs in Ghana 13 Years After National Policy Change.

PURPOSE: Malaria continues to be a major health issue globally with almost 85% of the global burden and deaths borne by sub-Saharan Africa and India. Although the current artemisinin derived combination therapies in Ghana are still efficacious against the Plasmodium falciparum (Pf) parasite, compounding evidence of artemisinin and amodiaquine resistance establish the need for a full, up-to-date understanding and monitoring of antimalarial resistance to provide evidence for planning control strategies. MATERIALS AND METHODS: The study was cross-sectional and was conducted during the peak malaria transmission seasons of 2015, 2016, and 2017 in two ecological zones of Ghana. Study participants included children aged 6 months to 14 years. Using ex vivo 4,6-diamidino-2-phenylindole (DAPI) drug sensitivity assay, 330 Pf isolates were used to investigate susceptibility to five antimalarial drugs: chloroquine (CQ), amodiaquine (AMD) dihydroartemisinin (DHA), artesunate (ART) and mefloquine (MFQ). RESULTS: The pooled geometric mean IC50S (GMIC50) of the five drugs against the parasites from Cape Coast and Begoro were 15.5, 42.4, 18.9, 4.6 and 27.3nM for CQ, AMD, DHA, ART, and MFQ, respectively. The GMIC50 values for CQ (p<0.001), ART (p<0.011) and DHA (p<0.018) were significantly higher for Cape Coast isolates as compared to Begoro isolates. However, GMIC50 estimates for MFQ (p<0.022) were significantly higher for Begoro isolates. Positive correlations were found between each pair of drugs with the weakest found between MFQ and DHA (r = 0.34;p<0.001), and the strongest between ART and DHA (r =0.66; p<0.001). CONCLUSION: The parasites showed reduced sensitivities to three (AMD, DHA and MFQ) out of the five drugs assessed. The study also demonstrated the continual return of chloroquine-sensitive parasites after 13 years of its withdrawal as the first-line drug for the treatment of uncomplicated malaria in Ghana. The ex vivo DAPI assay is a reliable method for assessing antimalarial drug sensitivities of Pf field isolates under field settings.

Histopathological lesions and exposure to Plasmodium falciparum infections in the placenta increases the risk of preeclampsia among pregnant women.

Preeclampsia (PE) is a placental disorder with different phenotypic presentations. In malaria-endemic regions, high incidence of PE is reported, with debilitating foeto-maternal effects, particularly among primigravid women. However, the relationship between placental pathology and Plasmodium falciparum infection in the placenta with PE is underexplored. Placentas from 134 pregnant women were examined after delivery for pathological lesions and placental malaria (PM). They comprised of 69 women without PE (non-PE group) and 65 women diagnosed with PE (PE group). The presence of placental pathology increased the risk of PE, with particular reference to syncytial knots. Placental malaria was 64 (48.1%) and 21 (15.8%) respectively for active and past infections and these proportions were significantly higher in the PE group compared to the non-PE group. Further multivariate analyses showed placental pathology (adjusted (aOR) 3.0, 95% CI = 1.2-7.5), active PM (aOR 6.7, 95% CI = 2.3-19.1), past PM (aOR 12.4, 95% CI = 3.0-51.0) and primigravidity (aOR 6.6, 95% CI 2.4-18.2) to be associated with PE. Our findings suggest that placental histological changes and PM are independent risk factors for PE particularly in primigravida. These findings might improve the management of PE in malaria-endemic regions.

Phenotypic Evidence of T Cell Exhaustion and Senescence During Symptomatic Plasmodium falciparum Malaria.

T cells play significant roles during Plasmodium falciparum infections. Their regulation of the immune response in symptomatic children with malaria has been deemed necessary to prevent immune associated pathology. In this study, we phenotypically characterized the expression of T cell inhibitory(PD-1, CTLA-4) and senescent markers (CD28(-), CD57) from children with symptomatic malaria, asymptomatic malaria and healthy controls using flow cytometry. We observed increased expression of T cell exhaustion and senescence markers in the symptomatic children compared to the asymptomatic and healthy controls. T cell senescence markers were more highly expressed on CD8 T cells than on CD4 T cells. Asymptomatically infected children had comparable levels of these markers with healthy controls except for CD8+ PD-1+ T cells which were significantly elevated in the asymptomatic children. Also, using multivariate regression analysis, CTLA-4 was the only marker that could predict parasitaemia level. The results suggest that the upregulation of immune exhaustion and senescence markers during symptomatic malaria may affect the effector function of T cells leading to inefficient clearance of parasites, hence the inability to develop sterile immunity to malaria.

Antigenicity and immune correlate assessment of seven Plasmodium falciparum antigens in a longitudinal infant cohort from northern Ghana.

The current global malaria control and elimination agenda requires development of additional effective disease intervention tools. Discovery and characterization of relevant parasite antigens is important for the development of new diagnostics and transmission monitoring tools and for subunit vaccine development. This study assessed the natural antibody response profile of seven novel Plasmodium falciparum pre-erythrocytic antigens and their potential association with protection against clinical malaria. Antigen-specific antibody levels in plasma collected at six time points from a longitudinal cohort of one-to-five year old children resident in a seasonal malaria transmission area of northern Ghana were assessed by ELISA. Antibody levels were compared between parasite-positive and parasite-negative individuals and the association of antibody levels with malaria risk assessed using a regression model. Plasma antibody levels against five of the seven antigens were significantly higher in parasite-positive children compared to parasite-negative children, especially during low transmission periods. None of the antigen-specific antibodies showed an association with protection against clinical malaria. The study identified five of the seven antigens as markers of exposure to malaria, and these will have relevance for the development of disease diagnostic and monitoring tools. The vaccine potential of these antigens requires further assessment.

Association between alpha-thalassaemia trait, Plasmodium falciparum asexual parasites and gametocyte carriage in a malaria endemic area in Southern Ghana.

OBJECTIVE: The alpha-thalassaemia trait has been associated with protection against severe malaria but its role in Plasmodium falciparum asexual parasite and gametocyte carriage remains unclear. This study examined association between prevalence of α-thalassaemia and P. falciparum asexual stage parasitaemia and gametocytaemia in children, pregnant women and adults, which was part of a bigger study that investigated some key factors that influence gametocyte carriage. RESULTS: Overall prevalence of heterozygous α-thalassaemia trait among all the groups was 39.0%, while 8.2% were homozygous alpha thalassaemia. Asexual parasite prevalence was significantly higher in children (P = 0.008) compared to adults and pregnant women. Of the asexual P. falciparum positive individuals, gametocyte prevalence was 38.5% (15/39) in children, 29.7% (11/37) in pregnant women and 17.4% (4/23) in adults. Heterozygous α-thalassaemic children were less likely to harbour asexual parasites, compared with normal and those deficient (OR = 0.52; 95% CI 0.28-0.97; P = 0.037) under the dominant model. These heterozygous children were also associated with reduced risk of parasitaemia compared to heterozygous adults and pregnant women. Children with heterozygous α-thalassaemia trait had reduced risk of asexual parasite carriage. There was however, no association between α-thalassaemia trait and risk of gametocyte carriage.

Novel Strategies for Malaria Vaccine Design.

The quest for a licensed effective vaccine against malaria remains a global priority. Even though classical vaccine design strategies have been successful for some viral and bacterial pathogens, little success has been achieved for Plasmodium falciparum, which causes the deadliest form of malaria due to its diversity and ability to evade host immune responses. Nevertheless, recent advances in vaccinology through high throughput discovery of immune correlates of protection, lymphocyte repertoire sequencing and structural design of immunogens, provide a comprehensive approach to identifying and designing a highly efficacious vaccine for malaria. In this review, we discuss novel vaccine approaches that can be employed in malaria vaccine design.

The prevalence of submicroscopic Plasmodium falciparum gametocyte carriage and multiplicity of infection in children, pregnant women and adults in a low malaria transmission area in Southern Ghana.

BACKGROUND: The gametocyte stage of Plasmodium falciparum is considered an important target for disrupting malaria transmission. Indications are that various demographic groups, such as children and pregnant women may differ in risk of harbouring gametocytes, which may be crucial for targeted control. In this study, the relationship between the prevalence and multiplicity of P. falciparum, asexual parasite infections and gametocytaemia was assessed in three different demographic groups in an area of southern Ghana with low malaria endemicity. Levels of antibody responses to Pfs230 were also assessed as a proxy for the presence of gametocytes. METHODS: The study involved multiple cross-sectional sampling of children (N = 184, aged 2-15 years), male and non-pregnant female adults (N = 154, aged 16-65 years) and pregnant women (N = 125, aged 18-45 years) from Asutsuare in the Shai Osudoku District of Greater Accra Region in Ghana. Asexual parasitaemia was detected by microscopy and PCR, and gametocytaemia was assessed by Pfs25-real time PCR. Multiclonal P. falciparum infections were estimated by msp2 genotyping and an indirect ELISA was used to measure plasma IgG antibodies to Pfs230 antigen. RESULTS: Overall, children and pregnant women had higher prevalence of submicroscopic gametocytes (39.5% and 29.7%, respectively) compared to adults (17.4%). Multiplicity of infection observed amongst children (3.1) and pregnant women (3.9) were found to be significantly higher (P = 0.006) compared with adults (2.7). Risk of gametocyte carriage was higher in individuals infected with P. falciparum having both Pfmsp2 3D7 and FC27 parasite types (OR = 5.92, 95% CI 1.56-22.54, P = 0.009) compared with those infected with only 3D7 or FC27 parasite types. In agreement with the parasite prevalence data, anti-Pfs230 antibody levels were lower in gametocyte positive adults (ß = - 0.57, 95% CI - 0.81, - 0.34, P < 0.001) compared to children. CONCLUSIONS: These findings suggest that children and pregnant women are particularly important as P. falciparum submicroscopic gametocyte reservoirs and represent important focus groups for control interventions. The number of clones increased in individuals carrying gametocytes compared to those who did not carry gametocytes. The higher anti-gametocyte antibody levels in children suggests recent exposure and may be a marker of gametocyte carriage.

Characterization of T cell activation and regulation in children with asymptomatic Plasmodium falciparum infection.

BACKGROUND: Asymptomatic Plasmodium infections are characterized by the absence of clinical disease and the ability to restrict parasite replication. Increasing levels of regulatory T cells (Tregs) in Plasmodium falciparum infections have been associated with the risk of developing clinical disease, suggesting that individuals with asymptomatic infections may have reduced Treg frequency. However, the relationship between Tregs, cellular activation and parasite control in asymptomatic malaria remains unclear. METHODS: In a cross-sectional study, the levels of Tregs and other T cell activation phenotypes were compared using flow cytometry in symptomatic, asymptomatic and uninfected children before and after stimulation with infected red blood cell lysates (iRBCs). In addition, the association between these T cell phenotypes and parasitaemia were investigated. RESULTS: In children with asymptomatic infections, levels of Tregs and activated T cells were comparable to those in healthy controls but significantly lower than those in symptomatic children. After iRBC stimulation, levels of Tregs remained lower for asymptomatic versus symptomatic children. In contrast, levels of activated T cells were higher for asymptomatic children. Strikingly, the pre-stimulation levels of two T cell activation phenotypes (CD8+CD69+ and CD8+CD25+CD69+) and the post-stimulation levels of two regulatory phenotypes (CD4+CD25+Foxp3+ and CD8+CD25+Foxp3+) were significantly positively correlated with and explained 68% of the individual variation in parasitaemia. A machine-learning model based on levels of these four phenotypes accurately distinguished between asymptomatic and symptomatic children (sensitivity = 86%, specificity = 94%), suggesting that these phenotypes govern the observed variation in disease status. CONCLUSION: Compared to symptomatic P. falciparum infections, in children asymptomatic infections are characterized by lower levels of Tregs and activated T cells, which are associated with lower parasitaemia. The results indicate that T cell regulatory and activation phenotypes govern both parasitaemia and disease status in paediatric malaria in the studied sub-Saharan African population.