Afucosylated Plasmodium falciparum-specific IgG is induced by infection but not by subunit vaccination.

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family members mediate receptor- and tissue-specific sequestration of infected erythrocytes (IEs) in malaria. Antibody responses are a central component of naturally acquired malaria immunity. PfEMP1-specific IgG likely protects by inhibiting IE sequestration and through IgG-Fc Receptor (FcγR) mediated phagocytosis and killing of antibody-opsonized IEs. The affinity of afucosylated IgG to FcγRIIIa is up to 40-fold higher than fucosylated IgG, resulting in enhanced antibody-dependent cellular cytotoxicity. Most IgG in plasma is fully fucosylated, but afucosylated IgG is elicited in response to enveloped viruses and to paternal alloantigens during pregnancy. Here we show that naturally acquired PfEMP1-specific IgG is strongly afucosylated in a stable and exposure-dependent manner, and efficiently induces FcγRIIIa-dependent natural killer (NK) cell degranulation. In contrast, immunization with a subunit PfEMP1 (VAR2CSA) vaccine results in fully fucosylated specific IgG. These results have implications for understanding protective natural- and vaccine-induced immunity to malaria.

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.

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.

Etiology of Placental Plasmodium falciparum Malaria in African Women.

Plasmodium falciparum parasites causing placental malaria express the VAR2CSA type of the clonally variant antigen family erythrocyte membrane protein 1 (PfEMP1). This enables evasion of preexisting immunity and results in placental accumulation of infected erythrocytes. We present data on seasonal variation in levels of VAR2CSA-specific immunoglobulin G (IgG) and IgG specific for a placental malaria-unrelated PfEMP1 protein among Ghanaian women at their first antenatal visit. Our results indicate that placental malaria does not require recent exposure to infected mosquitoes, in contrast to malaria in general. This has implications for the impact of insecticide-treated bed nets on placental malaria incidence and for antenatal care in woman with preexisting immunity.

Variations in the quality of malaria-specific antibodies with transmission intensity in a seasonal malaria transmission area of Northern Ghana.

INTRODUCTION: Plasmodium falciparum induced antibodies are key components of anti-malarial immunity in malaria endemic areas, but their antigen targets can be polymorphic. Induction of a high proportion of strain-specific antibodies will limit the recognition of a broad diversity of parasite strains by these responses. There are indications that circulating parasite diversity varies with malaria transmission intensity, and this may affect the specificity of elicited anti-malarial antibodies. This study therefore assessed the effect of varying malaria transmission patterns on the specificity of elicited antibody responses and to identify possible antibody correlates of naturally acquired immunity to malaria in children in an area of Ghana with seasonal malaria transmission. METHODS: This retrospective study utilized plasma samples collected longitudinally at six time points from children aged one to five years. Multiplex assays were used to measure antibody levels against four P. falciparum AMA 1 variants (from the 3D7, FVO, HB3 and CAMP parasite strains) and the 3D7 variant of the EBA 175 region II antigen and the levels compared between symptomatic and asymptomatic children. The relative proportions of cross-reactive and strain-specific antibodies against the four AMA 1 variants per sampling time point were assessed by Bland-Altman plots. The levels of antibodies against allelic AMA1 variants, measured by singleplex and multiplex luminex assays, were also compared. RESULTS: The data show that increased transmission intensity is associated with higher levels of cross-reactive antibody responses, most likely a result of a greater proportion of multiple parasite clone infections during the high transmission period. Anti-AMA1 antibodies were however associated with a history of infection rather than protection in this age group. CONCLUSION: The data contribute to understanding the underlying mechanism of the acquisition of strain-transcending antibody immunity following repeated exposure to diverse parasite strains.

Effect of IPTp on Plasmodium falciparum antibody levels among pregnant women and their babies in a sub-urban coastal area in Ghana.

BACKGROUND: Women exposed to Plasmodium infection develop antibodies and become semi-immune. This immunity is suppressed during pregnancy making both the pregnant woman and the foetus vulnerable to the adverse effects of malaria, particularly by Plasmodium falciparum. Intermittent preventive treatment of malaria in pregnancy (IPTp) with Sulfadoxine-pyrimethamine (SP) tablets is one of the current interventions to mitigate the effects of malaria on both the pregnant woman and the unborn child. The extent to which IPTp may interfere with the acquisition of protective immunity against pregnancy-associated malaria (PAM) is undefined in Ghana. METHODS: Three-hundred-and-twenty pregnant women were randomly enrolled at the antenatal clinic (ANC) in Madina, Accra. Venous blood samples were obtained at first ANC registration and at 4-week intervals (post-IPTp administration). Placental and cord blood samples were obtained at delivery and the infants were followed monthly for 6 months after birth. Anti-IgG and IgM antibodies against a crude antigen preparation and the glutamate-rich protein (GLURP) of P. falciparum were quantified by the enzyme-linked immunosorbent assay (ELISA). RESULTS: There was a general decline in the trend of mean concentrations of all the antibodies from enrolment to delivery. The levels of antibodies in cord blood and placenta were well correlated. Children did not show clinical signs of malaria at 6 months after birth. CONCLUSIONS: IgG against both crude antigen and GLURP were present in placenta and cord blood and it is therefore concluded that there is a trend of declining antibody from enrolment to delivery and IPTp-SP may have reduced malaria exposure, however, this does not impact on the transfer of antibodies to the foetus in utero. The levels of maternal and cord blood antibodies at delivery showed no adverse implications on malaria among the children at 6 months. However, the quantum and quality of the antibody transferred needs further investigation to ensure that the infants are protected from severe episodes of malaria.

Seroprevalence of Antibodies against Plasmodium falciparum Sporozoite Antigens as Predictive Disease Transmission Markers in an Area of Ghana with Seasonal Malaria Transmission.

INTRODUCTION: As an increasing number of malaria-endemic countries approach the disease elimination phase, sustenance of control efforts and effective monitoring are necessary to ensure success. Mathematical models that estimate anti-parasite antibody seroconversion rates are gaining relevance as more sensitive transmission intensity estimation tools. Models however estimate yearly seroconversion and seroreversion rates and usually predict long term changes in transmission, occurring years before the time of sampling. Another challenge is the identification of appropriate antigen targets since specific antibody levels must directly reflect changes in transmission patterns. We therefore investigated the potential of antibodies to sporozoite and blood stage antigens for detecting short term differences in malaria transmission in two communities in Northern Ghana with marked, seasonal transmission. METHODS: Cross-sectional surveys were conducted during the rainy and dry seasons in two communities, one in close proximity to an irrigation dam and the other at least 20 Km away from the dam. Antibodies against the sporozoite-specific antigens circumsporozoite protein (CSP) and Cell traversal for ookinetes and sporozoites (CelTOS) and the classical blood stage antigen apical membrane antigen 1 (AMA1) were measured by indirect ELISA. Antibody levels and seroprevalence were compared between surveys and between study communities. Antibody seroprevalence data were fitted to a modified reversible catalytic model to estimate the seroconversion and seroreversion rates. RESULTS: Changes in sporozoite-specific antibody levels and seroprevalence directly reflected differences in parasite prevalence between the rainy and dry seasons and hence the extent of malaria transmission. Seroconversion rate estimates from modelled seroprevalence data did not however support the above observation. CONCLUSIONS: The data confirms the potential utility of sporozoite-specific antigens as useful markers for monitoring short term/seasonal changes in malaria transmission. It may however be essential to update models to allow for assessment of seasonal changes in malaria transmission, which usually occur within four to six months.

Antibody levels against GLURP R2, MSP1 block 2 hybrid and AS202.11 and the risk of malaria in children living in hyperendemic (Burkina Faso) and hypo-endemic (Ghana) areas.

BACKGROUND: Differences in parasite transmission intensity influence the process of acquisition of host immunity to Plasmodium falciparum malaria and ultimately, the rate of malaria related morbidity and mortality. Potential vaccines being designed to complement current intervention efforts therefore need to be evaluated against different malaria endemicity backgrounds. METHODS: The associations between antibody responses to the chimeric merozoite surface protein 1 block 2 hybrid (MSP1 hybrid), glutamate-rich protein region 2 (GLURP R2) and the peptide AS202.11, and the risk of malaria were assessed in children living in malaria hyperendemic (Burkina Faso, n = 354) and hypo-endemic (Ghana, n = 209) areas. Using the same reagent lots and standardized protocols for both study sites, immunoglobulin (Ig) M, IgG and IgG sub-class levels to each antigen were measured by ELISA in plasma from the children (aged 6-72 months). Associations between antibody levels and risk of malaria were assessed using Cox regression models adjusting for covariates. RESULTS: There was a significant association between GLURP R2 IgG3 and reduced risk of malaria after adjusting age of children in both the Burkinabe (hazard ratio 0.82; 95 % CI 0.74-0.91, p < 0.0001) and the Ghanaian (HR 0.48; 95 % CI 0.25-0.91, p = 0.02) cohorts. MSP1 hybrid IgM was associated (HR 0.85; 95 % CI 0.73-0.98, p = 0.02) with reduced risk of malaria in Burkina Faso cohort while IgG against AS202.11 in the Ghanaian children was associated with increased risk of malaria (HR 1.29; 95 % CI 1.01-1.65, p = 0.04). CONCLUSION: These findings support further development of GLURP R2 and MSP1 block 2 hybrid, perhaps as a fusion vaccine antigen targeting malaria blood stage that can be deployed in areas of varying transmission intensity.

Analysis of Erythrocyte Invasion Mechanisms of Plasmodium falciparum Clinical Isolates Across 3 Malaria-Endemic Areas in Ghana.

BACKGROUND: Plasmodium falciparum invades human erythrocytes by using an array of ligands that interact with several receptors, including sialic acid (SA), complement receptor 1 (CR1), and basigin. We hypothesized that in malaria-endemic areas, parasites vary invasion pathways under immune pressure. Therefore, invasion mechanisms of clinical isolates collected from 3 zones of Ghana with different levels of endemicity (from lowest to highest, Accra, Navrongo, and Kintampo) were compared using standardized methods. METHODS: Blood samples were collected from children aged 2-14 years in whom malaria was diagnosed, and erythrocyte invasion phenotypes were determined using the enzymes neuraminidase, chymotrypsin, and trypsin, which differentially cleave receptors from the erythrocyte surface. In addition, antibodies against CR1 and basigin were used to determine the contributions of these receptors to invasion. Gene expression levels of P. falciparum invasion ligands were also examined. RESULTS: The parasites generally expressed SA-independent invasion phenotypes across the malaria-endemic areas, with parasites from Kintampo showing the highest invasion rates in neuraminidase-treated erythrocytes. CR1 was a major mediator of SA-independent invasion, while basigin was essential for both SA-dependent and SA-independent invasion mechanisms. Furthermore, expression of the basigin ligand PfRh5 was the best predictor of donor parasitemia. CONCLUSIONS: Erythrocyte invasion phenotypes expressed by P. falciparum are influenced by endemicity levels, and the PfRh5-basigin pathway is a potential vaccine target.

Anti-sporozoite antibodies as alternative markers for malaria transmission intensity estimation.

BACKGROUND: Reported malaria cases continue to decline globally, and this has been attributed to strategic implementation of multiple malaria control tools. Gains made would however need to be sustained through continuous monitoring to ensure malaria elimination and eradication. Entomological inoculation rate (EIR) is currently the standard tool for transmission monitoring but this is not sensitive enough, especially in areas of very low transmission. Transmission estimation models based on seroconversion rates (λ) of antibodies to Plasmodium falciparum blood stage antigens are gaining relevance. Estimates of λ, which is the measure of transmission intensity, correlate with EIR but are limited by long-term persistence of antibodies to blood stage antigens. Seroprevalence of antibodies to sporozoite antigens may be better alternatives since these antigens usually have shorter immune exposure times. The aim of this study was to develop transmission estimation models based on the seroprevalence of antibodies to two P. falciparum sporozoite antigens (CSP, CelTOS) and compare with models based on the classical blood stage antigen AMA1. METHODS: Antibody levels in archived plasma from three cross-sectional surveys conducted in 2009 in a low transmission area of Southern Ghana were assessed by indirect ELISA. Seroprevalence of antibodies against CSP, CelTOS and AMA1 were fitted to reversible catalytic models to estimate λ and corresponding seroreversion rates (ρ) for each antibody. RESULTS: Of the three models developed, the anti-CSP model predicted a 13-fold decrease in λ four years prior to the time of sampling (2009). Anti-AMA1 antibodies formed at a four-fold greater rate compared to that of anti-CelTOS antibodies, and anti-CSP antibodies during the period of decreased λ. In contrast, anti-AMA1 antibodies decayed at a five-fold slower rate relative to that of anti-CSP antibodies while anti-AMA1 and anti-CelTOS antibody decay rates were not significantly different. Anti-CSP antibodies were relatively short-lived as they formed at an 11.6-fold slower rate relative to their decay during the period of decreased λ. CONCLUSIONS: These features of anti-CSP antibodies can be exploited for the development of models for predicting seasonal, short-term changes in transmission intensity in malaria-endemic areas, especially as the elimination phase of malaria control is approached.

Recombinant ESAT-6-CFP10 Fusion Protein Induction of Th1/Th2 Cytokines and FoxP3 Expressing Treg Cells in Pulmonary TB.

BACKGROUND: Early secretory antigenic target 6 (ESAT-6) and culture filtrate protein 10 (CFP-10) are Mycobacterium tuberculosis (Mtb)-specific antigens that are secreted by actively metabolising bacteria and contribute to the virulence of the bacteria. Their ability to induce Treg and Th2 responses, particularly during the first two weeks of treatment, has not been comprehensively examined to date. The purpose of this work was to characterise Th1, Th2 and Treg responses to rESAT-6-CFP10 fusion protein in TB patients before and during the intensive phase of treatment and in healthy M.bovis BCG vaccinated donors. METHODS: Forty-six newly diagnosed, HIV-negative, smear-positive pulmonary TB patients and 20 healthy donors were recruited in the UK and Ghana. Their peripheral blood mononuclear cells (PBMC) were used in ex vivo ELISPOT and in vitro cultures to identify immunological parameters of interest. RESULTS: The study confirmed that protective immune responses to rESAT-6-CFP10 are impaired in active TB but improved during treatment: circulating antigen-specific IL-4-producing T-cells were increased in untreated TB but declined by two weeks of treatment while the circulating antigen-specific IFN-γ producing T cells which showed a transient rise at one week of treatment, persisted at baseline levels at two months of treatment. In vitro T cell proliferation and IFN-γ production were reduced, while IL-4 and CD4(+)FoxP3(+)CD25(hi) cell expression were increased in response to rESAT-6-CFP10 fusion protein in untreated TB. These responses were reversed during early treatment of TB. CONCLUSIONS: These observations support further investigations into the possible utility of these parameters as markers of active disease and favourable treatment outcomes.