Histidine-rich Protein Research Articles

A Novel Malaria Lateral Flow Assay for Detecting Plasmodium falciparum Lactate Dehydrogenase in Busia, Uganda.

ABSTRACT.Rapid diagnostic tests (RDTs) for Plasmodium falciparum commonly detect histidine-rich protein 2 (HRP-2), but HRP-2 deletions are increasingly recognized. We evaluated a prototype test detecting parasite lactate dehydrogenase (pLDH) and compared it to commercially available RDTs at a health facility in Uganda, using quantitative polymerase chain reaction as a gold standard. The prototype pLDH test had a high sensitivity for infections with at least 100 parasites/µL (98%), comparable to HRP-2, and greater than an existing pLDH RDT (89%). Specificity for the prototype test was 99.5%, which is greater than the HRP-2 tests (93–95%). Therefore, the prototype pLDH test may be an attractive alternative malaria diagnostic.

Modelling the epidemiology of malaria and spread of HRP2-negative Plasmodium falciparum following the replacement of HRP2-detecting rapid diagnostic tests.

Malaria rapid diagnostic tests (RDTs) are dominated by products which use histidine-rich protein 2 (HRP2) to detect Plasmodium falciparum. The emergence of parasites lacking the pfhrp2 gene can lead to high rates of false-negative results amongst these RDTs. One solution to restore the ability to correctly diagnose falciparum malaria is to switch to an RDT which is not solely reliant on HRP2. This study used an agent-based stochastic simulation model to investigate the impact on prevalence and transmission caused by switching the type of RDT used once false-negative rates reached pre-defined thresholds within the treatment-seeking symptomatic population. The results show that low transmission settings were the first to reach the false-negative switch threshold, and that lower thresholds were typically associated with better long-term outcomes. Changing the diagnostic RDT away from a HRP2-only RDT is predicted to restore the ability to correctly diagnose symptomatic malaria infections, but often did not lead to the extinction of HRP2-negative parasites from the population which continued to circulate in low density infections, or return to the parasite prevalence and transmission levels seen prior to the introduction of the HRP2-negative parasite. In contrast, failure to move away from HRP2-only RDTs leads to near fixation of these parasites in the population, and the inability to correctly diagnose symptomatic cases. Overall, these results suggest pfhrp2-deleted parasites are likely to become a significant component of P. falciparum parasite populations, and that long-term strategies are needed for diagnosis and surveillance which do not rely solely on HRP2.

Plasmodium falciparum histidine rich protein 2 (pfhrp2): an additional genetic marker suitable for anti-malarial drug efficacy trials

BackgroundGenotyping of the three Plasmodium falciparum polymorphic genes, msp1, msp2 and glurp, has been adopted as a standard strategy to distinguish recrudescence from new infection in drug efficacy clinical trials. However, the suitability of a particular gene is compromised in areas where its allelic variants distribution is significantly skewed, a phenomenon that might occur in isolated parasite populations or in areas of very low transmission. Moreover, observation of amplification bias has diminished the value of glurp as a marker.MethodsThe suitability of the polymorphic P. falciparum histidine-rich protein 2 (pfhrp2) gene was assessed to serve as an alternative marker using a PCR-sequencing or a PCR–RFLP protocol for genotyping of samples in drug efficacy clinical trials. The value of pfhrp2 was validated by side-by-side analyses of 5 admission-recrudescence sample pairs from Yemeni malaria patients.ResultsThe outcome of the single pfhrp2 gene discrimination analysis has been found consistent with msp1, msp2 and glurp pool genotyping analysis for the differentiation of recrudescence from new infection.ConclusionThe findings suggest that under the appropriate circumstances, pfhrp2 can serve as an additional molecular marker for monitoring anti-malarials efficacy. However, its use is restricted to endemic areas where only a minority of P. falciparum parasites lack the pfhrp2 gene.

Histidine-rich calcium binding protein promotes gastric cancer cell proliferation, migration, invasion and epithelial-mesenchymal transition through Raf/MEK/ERK signaling.

Histidine-rich calcium binding protein (HRC) is a new type of Ca2+ homeostasis regulator, which acts as a nonnegligible role in regulating intracellular calcium homeostasis. Here, we demonstrated that HRC expression was upregulated in human gastric cancer (GC) samples, and its expression level was closely correlated with the overall survival (OS) rate of GC patients and the malignant potential of GC cell lines. Knockdown of HRC inhibited migration, invasion, and proliferation of GC cell lines in vitro, while HRC overexpression promoted GC cell migration, invasion, and proliferation in vitro, as well as the growth of subcutaneous tumors and peritoneal tumors in vivo. In terms of the mechanism, knockdown of HRC reduced the intracellular calcium ion level and the CaM protein level. Through cell function experiments, we found that HRC regulated the Raf/MEK/ERK pathway through Ca2+/CaM signaling and ultimately affected the epithelial‑mesenchyme transition (EMT) of GC. In summary, we revealed that HRC represents a potential target for GC treatment.

Malaria hemozoin: A target for point-of-care diagnosis.

The challenges in malaria diagnosis continue to threaten the malaria elimination goal in India and other malaria-endemic countries. A rapid diagnostic test (RDT) kit is widely used in resource-constrained areas where microscopy and molecular methods are not easily deployable. Considering the problems associated with the currently available RDT kit, such as histidine-rich protein 2 gene deletion and prolonged stability of the protein in the blood, it suggests that new potential biomarkers are urgently needed. Hemozoin (Hz) is an important biomarker for malaria diagnosis, which is the by-product of a detoxification mechanism in the malaria parasite. This article highlights the importance of "Hz" for point-of-care malaria diagnosis when India and other countries are moving toward the goal of malaria elimination.

Prevalence of Malaria in Apparently Healthy Healthcare Workers in Dass Local Government Area, Bauchi State, Nigeria

Background: Malaria is among the world’s life-threatening protozoan diseases, especially in the tropics with increasing morbidity and mortality at an unprecedented high level. Healthcare workers (HCWs) in the tropics are increasingly affected despite the availability of effective preventive interventions. The study determines the prevalence of malarial parasitaemia in apparently healthy HCWs in Dass Local Government Area of Bauchi State. Methods: A schedule was used to collect information from 223 HCWs using a cross-sectional study design. The respondents were selected through multistage sampling. Blood sample from each participant was collected for malaria diagnosis using both Microscopy and Rapid Diagnostic Test (P. falciparum histidine-rich protein-2, HRP-2 mRDT). Results: The prevalence of malaria was 55.2 % using Microscopy (HRP-2 mRDT- 40.4 %). The RDT kit has a sensitivity of 66.7 %, specificity of 92.0 %, positive predictive value (PPV) of 91.1 % and a negative predictive value (NPV) of 69.2 %. The majority of the positive cases (91.9 %) had low parasitaemia (< 1,000 parasites per µL of blood ) and 8.1 % had moderate parasitaemia (1,000 - 9,999 parasites per µL of blood). A significant association (χ² = 78. 868, df = 1, p < 0.001, phi = 0.595) exists between RDT and Microscopy with a large effect size. No significant association between asymptomatic parasitaemia and insecticide-treated nets (ITNs) utilization (p ≥ 0.05). Conclusions: The prevalence of asymptomatic malarial parasitaemia was high despite the use of ITNs suggesting workplace hazard-related exposure. In addition to ITNs use, proper environmental sanitation (ensuring proper drainage systems), use of long-sleeved stockings, mosquito repellents and mosquito sprays should be encouraged in health facilities.

Prevalence of Malaria in Apparently Healthy Healthcare Workers in Dass Local Government Area, Bauchi State, Nigeria

Background: Malaria is among the world’s life-threatening protozoan diseases, especially in the tropics with increasing morbidity and mortality at an unprecedented high level. Healthcare workers (HCWs) in the tropics are increasingly affected despite the availability of effective preventive interventions. The study determines the prevalence of malarial parasitaemia in apparently healthy HCWs in Dass Local Government Area of Bauchi State. Methods: A schedule was used to collect information from 223 HCWs using a cross-sectional study design. The respondents were selected through multistage sampling. Blood sample from each participant was collected for malaria diagnosis using both Microscopy and Rapid Diagnostic Test (P. falciparum histidine-rich protein-2, HRP-2 mRDT). Results: The prevalence of malaria was 55.2 % using Microscopy (HRP-2 mRDT- 40.4 %). The RDT kit has a sensitivity of 66.7 %, specificity of 92.0 %, positive predictive value (PPV) of 91.1 % and a negative predictive value (NPV) of 69.2 %. The majority of the positive cases (91.9 %) had low parasitaemia (< 1,000 parasites per µL of blood ) and 8.1 % had moderate parasitaemia (1,000 - 9,999 parasites per µL of blood). A significant association (χ² = 78. 868, df = 1, p < 0.001, phi = 0.595) exists between RDT and Microscopy with a large effect size. No significant association between asymptomatic parasitaemia and insecticide-treated nets (ITNs) utilization (p ≥ 0.05). Conclusions: The prevalence of asymptomatic malarial parasitaemia was high despite the use of ITNs suggesting workplace hazard-related exposure. In addition to ITNs use, proper environmental sanitation (ensuring proper drainage systems), use of long-sleeved stockings, mosquito repellents and mosquito sprays should be encouraged in health facilities.

MoO3-templated synthesis of TiO2@C-Ni microtubes for efficient catalysis and protein adsorption

The hybrid structure of metallic Ni nanoparticles (NPs)-decorated micro/nanotubes has the merits of both unique property of Ni NPs and tubular structure of micro/nanotubes, which is highly desired for many applications. In this study, Ni NPs decorated porous TiO2 microtubes with N-doped carbon layer from polydopamine (PDA) are directly synthesized by an extend stöber method, mussel chemistry and subsequent annealing treatment for the first time. In the synthetic process, a shell layer of nickel ion-embedded in polydopamine (PDA-Ni2+) was covered on the MoO3@TiO2 microcables by nickel ion mediated dopamine polymerization in ammonia solution, resulting in the formation of amorphous TiO2@PDA-Ni2+ microtubes. Notably, the coating of PDA-Ni2+ layer and the dissolution of the MoO3 cores were easily achieved within one-step reaction, greatly facilitating the synthetic progress. After being annealed in nitrogen atmosphere at different temperature, the TiO2@C-Ni microtubes with anatase, rutile phase of TiO2 can be obtained. Benefiting from the high coverage of Ni NPs, large specific surface area as well as one dimensional tubular structure, the as-prepared TiO2@C-Ni microtubes exhibited excellent performance on adsorption for histidine-rich proteins as well as the reduction of 4-nitrophenol. These results may pave a new avenue on constructing 1D TiO2-based microtubes for potential applications in energy storage, photocatalysis etc.

In vitro transcytosis of Helicobacter pylori histidine-rich protein through gastric epithelial-like cells and the blood-brain barrier.

Recent epidemiological studies have supported the correlation between Helicobacter pylori infection and the development of Alzheimer's disease. HpHpn, a histidine-rich H. pylori protein, forms amyloid-like oligomers; it may be a pathogenic factor for Alzheimer's disease progression. HpHpn may also be transported from the gastric epithelium to the brain. However, HpHpn is secreted from H. pylori on the outer surface of gastric epithelia; therefore, the hypothesized movement of HpHpn across the gastric epithelium to the blood remains controversial. Here, we found the HpHpn showed acidic pH-dependent cellular uptake and subsequent secretion in human gastric epithelial-like carcinoma cells. Furthermore, HpHpn exhibited in vitro permeability across the blood-brain barrier. Although further in vivo experiments are required, our findings suggest that in vitro transcytosis of HpHpn in gastric epithelial cells and the blood-brain barrier may provide new insights into the correlation between H. pylori infections and Alzheimer's disease progression.

Prevalence of Infection and Malaria Parasite Density among Under Five Children: A Case Study of Dunukofia Rural Community in Anambra State, Nigeria

Aim: Malaria still remains an overwhelming cause of morbidity and mortality among children under five years of age, especially in sub-Saharan Africa. The study was carried out to evaluate malaria prevalence amongst children less than five years old. 
 Study Design: A cross sectional study was carried out. The study adopted a retrospective descriptive survey using the hospital records and diagnostic cross sectional survey by examination of blood samples across three variables: gender, age group and mosquito net usage.
 Duration: The study was done in 2021 from the month of March to April in the rural community. 
 Methodology: Parasitological diagnosis was with Plasmodium falciparum histidine-rich protein 2-based malaria Rapid Diagnostic Test (RDT) and microscopy of giemsa-stained blood smears. Demographic information was collected using questionnaire.
 Results: Three hundred (300) children aged less than five years malaria infection status was investigate, 174 (58.00% ) of them were females while 126 (42.00%. ) were males. Twenty one percent (21.00%) of the respondents are <1 year, 23.33% (70) of them are between the ages of 2 to 3 years, while 55.67% (167) were 4 years and above. Current malaria prevalence was higher with microscopy (67.33%) than that of RDT (23.33%). Also, previous RDT results showed that there was a higher prevalence (73.56%) of malaria parasites in females than males (58.73%). The microscopy results showed that males had a higher prevalence (38.10%) of malaria parasites than females (12.64%). Overall gender result also revealed that males had a higher prevalence (96.83%) of malaria parasites than females (86.21%). There was a significant difference in the prevalence result with gender (P<0.05). Females had higher parasite density (28.05±15.390) than males (23.22±19.171), there was no significant difference (P>0.05). It further revealed that children from 4 years and above had higher intensity (29.68±17.357) while those of 1 year and below had the least (14.89±16.069). However, there was no significant difference in the malaria parasite among the age groups of patients (P>0.05).
 Conclusion: Prevalence of malaria parasitaemia was still high in Dunukaofia, Anamba State, Nigeria despite various control measures and interventions put in place by WHO.

Limited Polymorphism in Plasmodium falciparum Artemisinin Resistance Kelch13-Propeller Gene Among Clinical Isolates from Bushenyi District, Uganda.

IntroductionDrug resistance remains a major challenge in malaria treatment, especially after the emergence of resistance to artemisinin-based combined therapies. Plasmodium falciparum Kelch13 gene mutations are implicated in conferring artemisinin resistance. Thus, this study was aimed at determining the occurrence of Kelch13 (K13) propeller resistance gene polymorphism mutations in Bushenyi district, Uganda.MethodsParticipants suspected to have malaria were recruited. P. falciparum was confirmed using antigen histidine-rich protein 2 (HRP2) (Pf) (Access Bio, Inc, USA) and microscopy. Malaria-positive patients were treated with artemeter-lumefantrine (AL). Blood was withdrawn from participants who tested positive for parasites after day 3 and kept in blood filter papers (ET31CHR; Whatman Limited, Kent, UK). DNA was extracted using chelex-suspension method. Nested polymerase chain reaction (PCR) was conducted and the second-round products sequenced using Sanger’s method. Sequenced products were analyzed using DNAsp 5.10.01 software and then blasted on to the NCBI for K13-propeller gene sequence identity using the Basic Local Alignment Search Tool (BLAST).ResultsOut of 283 enrolled participants, 194 completed the follow-up schedule. A total of 134 (69%) had no parasites on day 3, while 60 (31%) had parasites on that day. Out of the 60 samples, 40 (62%) were positively amplified as P. falciparum, with polymorphisms in the K13-propeller gene detected in 3 (7.5%) out of the 40 amplicons. Polymorphisms at codon 1929, 1788 and 1801 were detected separately in one sample each. Sequences have been deposited in NCBI with accession numbers PRJNA720348 and PRJNA720800.ConclusionPolymorphisms in the K13-propeller gene previously reported to be associated with artemisinin resistance were not detected in the P. falciparum isolates from Bushenyi district, Uganda. More studies need to be conducted on the new mutations detected so as to understand their association, if any, with ACT resistance.

A Ricin B-Like Lectin Protein Physically Interacts with TaPFT and Is Involved in Resistance to Fusarium Head Blight in Wheat.

Fusarium head blight (FHB), mainly caused by Fusarium graminearum, has become one of the most serious diseases that damage wheat. The TaPFT (pore-forming toxin-like) and TaHRC (histidine-rich calcium-binding protein) genes at the quantitative trait locus Fhb1 were identified to confer resistance to FHB in the wheat cultivar Sumai 3. In this study, a wheat ricin B-like lectin gene (designated TaRBL) that interacted with TaPFT was isolated by a yeast two-hybrid screen of a wheat cDNA library. A yeast two-hybrid and bimolecular fluorescence complementation study further verified that TaRBL interacted with TaPFT but not with TaHRC. Gene expression studies showed that upon F. graminearum infection, TaRBL expression was upregulated in resistant cultivars but downregulated in susceptible cultivars. Furthermore, knockdown of TaRBL expression by barley stripe mosaic virus-induced gene silencing significantly reduced the resistance of wheat to FHB in both the resistant cultivar Sumai 3 and the susceptible cultivar Jimai 22. Thus, we conclude that TaRBL encodes a ricin B-like lectin protein that interacts with TaPFT and is involved in resistance to FHB in wheat.

Missed Plasmodium falciparum and Plasmodium vivax Mixed Infections in Ethiopia Threaten Malaria Elimination.

ABSTRACT.Plasmodium falciparum and Plasmodium vivax are co-endemic in Ethiopia. This study investigated whether mixed infections were missed by microscopy from a 2017 therapeutic efficacy study at two health facilities in Ethiopia. All patients (N = 304) were initially classified as having single-species P. falciparum (n = 148 samples) or P. vivax infections (n = 156). Dried blood spots were tested for Plasmodium antigens by bead-based multiplex assay for pan-Plasmodium aldolase, pan-Plasmodium lactate dehydrogenase, P. vivax lactate dehydrogenase, and histidine-rich protein 2. Of 304 blood samples, 13 (4.3%) contained both P. falciparum and P. vivax antigens and were analyzed by polymerase chain reaction for species-specific DNA. Of these 13 samples, five were confirmed by polymerase chain reaction for P. falciparum/P. vivax co-infection. One sample, initially classified as P. vivax by microscopy, was found to only have Plasmodium ovale DNA. Plasmodium falciparum/P. vivax mixed infections can be missed by microscopy even in the context of a therapeutic efficacy study with multiple trained readers.

Hepatitis B and Asymptomatic Malaria Infection among Pregnant Women in a Semiurban Community of North-Central Nigeria.

Background The overlap of malaria and Hepatitis B Virus (HBV) infections present a major threat to public health throughout endemic countries of tropical and sub-Saharan Africa. There is a paucity of data on the prevalence and associated factors of malaria and HBV infections among pregnant women in Ejule, a semiurban area of Nigeria. Therefore, the current study was designed to assess the seroprevalence of malaria and HBV among pregnant women attending antenatal clinics in Ejule Metropolis. Materials and Methods In a hospital-based cross-sectional study, blood samples collected from 200 apparently healthy pregnant women at the Ilemona Clinic were screened for Plasmodium falciparum (P. falciparum) and HBsAg using histidine-rich protein 2 (HRP2) and hepatitis B surface antigen (HBsAg) rapid diagnostic tests (RDTs), respectively. Relevant sociodemographic and putative risk factor information was obtained with structured questionnaires. Results The prevalence of the infections was 44 (22%), 5 (2.5%), and 1 (0.5%) for P. falciparum monoinfection and HBV monoinfection and coinfection, respectively. Single and concurrent infections peaked at ages 31–40 years but decreased with older ages. High P. falciparum, 31 (59.62%), and HBV 2 (3.85%) infection were observed among those without formal education. Contrary to ages, occupation, and knowledge of infection, malaria parasitemia differed significantly with lower educational qualification (p ≤ 0.001), being single (p=0.001), and inconsistent use of insecticide-treated bed nets (ITNs) (p=0.04, OR = 5, CI: 0.10–0.47). History of blood donation (OR = 5, p=0.04, CI: 1.10–32.80) and multiple sex partners (OR = 11.9, p=0.01, CI: 0.01–0.93) were found to be significantly associated with hepatitis B surface antigenemia rate during pregnancy. No evidence of HBV infection was observed in women with a history of HBV vaccination. Conclusions Malaria is still highly prevalent among pregnant women due to high illiteracy and noncompliance to using ITNs. Therefore, routine screening and educating pregnant mothers are crucial in eliminating malaria in endemic settings. The low rate of hepatitis B and coinfection with malaria shows that further improvement in HBV vaccination could considerably reduce the disease burden among pregnant women.

Fabrication of Yolk-Shell Fe3O4@NiSiO3/Ni Microspheres for Efficient Purification of Histidine-Rich Proteins.

Magnetic materials perform well in the purification of histidine-rich proteins (His-proteins). In this work, a facile fabrication of yolk-shell magnetic Fe3O4@NiSiO3/Ni microspheres for the efficient purification of His-proteins has been reported. Yolk-shell Fe3O4@NiSiO3 microspheres were prepared via hydrothermal reaction. Then Ni nanoparticles (NPs) were loaded on Fe3O4@NiSiO3 microspheres after the adsorption and in situ reduction of nickel acetylacetonate. The yolk-shell Fe3O4@NiSiO3/Ni microspheres had a hierarchical flower-like structure and large cavities. The size of the cavity depended on the reaction time. This indicated that the microspheres had a large specific surface area for loading of more Ni NPs, which was crucial to the high His-protein adsorption capacity of Fe3O4@NiSiO3/Ni microspheres. Fe3O4@NiSiO3/Ni microspheres had a high adsorption capacity for bovine hemoglobin (BHb, 2822 mg/g), which was better than the values of other His-protein adsorbents. Fe3O4@NiSiO3/Ni microspheres still had a high BHb separation efficiency after seven separation cycles, indicating its good reusability and stability. Therefore, the as-prepared bifunctional yolk-shell Fe3O4@NiSiO3/Ni microspheres exhibited great practical application value for His-protein purification.

Plasmodium falciparum pfhrp2 and pfhrp3 gene deletions among patients in the DRC enrolled from 2017 to 2018

Rapid diagnostic tests (RDTs) detecting histidine-rich protein 2 (HRP2) and HRP3 are widely used throughout sub-Saharan Africa (SSA) to diagnose Plasmodium falciparum malaria. However, multiple SSA countries have reported pfhrp2 and pfhrp3 (pfhrp2/3) gene deletions. Blood samples (n = 1109) collected from patients with P. falciparum infection from six health facilities throughout the Democratic Republic of the Congo (DRC) from March 2017 to January 2018 were evaluated for pfhrp2/3 deletions. Samples were assayed for HRP2, pan-Plasmodium LDH (pLDH) and aldolase (pAldolase) antigens by bead-based multiplex antigen assay. Samples with low HRP2 concentration compared to pLDH and pAldolase antigens were selected for further pfhrp2/3 genotyping PCRs. The majority of blood samples (93.3%, 1035/1109) had high concentrations of the HRP2 antigen. Single deletions of pfhrp2 were identified in 0.27% (3/1109) of screened samples, with one sample from each of the Kapolowe, Mikalayi, and Rutshuru study sites. A pfhrp3 single deletion (0.09%, 1/1109) was found in the Kapolowe site. Dual pfhrp2 and pfhrp3 deletions were not observed. Due to, the low numbers of pfhrp2 deletions and the sporadic locations of these deletions, the use of HRP2-based RDTs appears to still be appropriate for these locations in DRC.

Performance of a Histidine Rich Protein-2 Based (First Response) and a p-Lactate Dehydrogenase-based (Optimal) Rapid Diagnostic Test for Diagnosis of Malaria in Patients With Pediatric Sickle Cell Disease.

Rapid diagnostic tests (RDTs) have been extensively evaluated and play an important role in malaria diagnosis. However, the accuracy of RDTs for malaria diagnosis in patients with sickle cell disease (SCD) is unknown. We compared the performance of a histidine rich protein 2 (HRP-2)-based RDT (First Response) and a lactate dehydrogenase (LDH)-based RDT (Optimal) with routine microscopy as reference standard in 445 children with SCD and an acute febrile illness in Accra, Ghana. The overall sensitivity, specificity, and positive and negative predictive values of the HRP-2-based RDTs were 100%, 95.7%, 73.8%, and 100%, respectively. Comparable values for the LDH-based RDTs were 91.7%, 99.5%, 95.7%, and 99.0%, respectively. A total of 423 results were true in both tests, 1 result was false in both tests, 16 results were false in the HRP-2 test only, and 5 were false in the LDH test only (McNemar test, P = .03). At follow-up, 73.7% (28/38), 52.6% (20/38), 48.6% (17/35), and 13.2% (5/38) of study participants were HRP-2 positive on days 14, 28, 35, and 42, respectively, compared with 0%, 2.6% (1/38), 2.9% (1/35), and 2.6% (1/38) for LDH. The HRP2-based RDT fulfilled World Health Organization criteria for malaria diagnosis in patients with SCD and may provide diagnostic evidence for treatment to begin in cases in which treatment would otherwise have begun presumptively based on symptoms, whereas LDH-based RDTs may be more suitable as a confirmatory test in low-parasitemic subgroups, such as patients with SCD.

Safety, infectivity and immunogenicity of a genetically attenuated blood-stage malaria vaccine

BackgroundThere is a clear need for novel approaches to malaria vaccine development. We aimed to develop a genetically attenuated blood-stage vaccine and test its safety, infectivity, and immunogenicity in healthy volunteers. Our approach was to target the gene encoding the knob-associated histidine-rich protein (KAHRP), which is responsible for the assembly of knob structures at the infected erythrocyte surface. Knobs are required for correct display of the polymorphic adhesion ligand P. falciparum erythrocyte membrane protein 1 (PfEMP1), a key virulence determinant encoded by a repertoire of var genes.MethodsThe gene encoding KAHRP was deleted from P. falciparum 3D7 and a master cell bank was produced in accordance with Good Manufacturing Practice. Eight malaria naïve males were intravenously inoculated (day 0) with 1800 (2 subjects), 1.8 × 105 (2 subjects), or 3 × 106 viable parasites (4 subjects). Parasitemia was measured using qPCR; immunogenicity was determined using standard assays. Parasites were rescued into culture for in vitro analyses (genome sequencing, cytoadhesion assays, scanning electron microscopy, var gene expression).ResultsNone of the subjects who were administered with 1800 or 1.8 × 105 parasites developed parasitemia; 3/4 subjects administered 3× 106 parasites developed significant parasitemia, first detected on days 13, 18, and 22. One of these three subjects developed symptoms of malaria simultaneously with influenza B (day 17; 14,022 parasites/mL); one subject developed mild symptoms on day 28 (19,956 parasites/mL); and one subject remained asymptomatic up to day 35 (5046 parasites/mL). Parasitemia rapidly cleared with artemether/lumefantrine. Parasitemia induced a parasite-specific antibody and cell-mediated immune response. Parasites cultured ex vivo exhibited genotypic and phenotypic properties similar to inoculated parasites, although the var gene expression profile changed during growth in vivo.ConclusionsThis study represents the first clinical investigation of a genetically attenuated blood-stage human malaria vaccine. A P. falciparum 3D7 kahrp– strain was tested in vivo and found to be immunogenic but can lead to patent parasitemia at high doses.Trial registrationAustralian New Zealand Clinical Trials Registry (number: ACTRN12617000824369; date: 06 June 2017).

Antithrombin protects against Plasmodium falciparum histidine-rich protein II-mediated inflammation and coagulation

AbstractPlasmodium falciparum-derived histidine-rich protein II (HRPII) has been shown to inhibit heparin-dependent anticoagulant activity of antithrombin (AT) and induce inflammation in vitro and in vivo. In a recent study, we showed that HRPII interacts with the AT-binding vascular glycosaminoglycans (GAGs) not only to disrupt the barrier-permeability function of endothelial cells but also to inhibit the antiinflammatory signaling function of AT. Here we investigated the mechanisms of the proinflammatory function of HRPII and the protective activity of AT in cellular and animal models. We found that AT competitively inhibits the GAG-dependent HRPII-mediated activation of NF-κB and expression of intercellular cell adhesion molecule 1 (ICAM1) in endothelial cells. Furthermore, AT inhibits HRPII-mediated histone H3 citrullination and neutrophil extracellular trap (NET) formation in HL60 cells and freshly isolated human neutrophils. In vivo, HRPII induced Mac1 expression on blood neutrophils, MPO release in plasma, neutrophil infiltration, and histone H3 citrullination in the lung tissues. HRPII also induced endothelial cell activation as measured by increased ICAM1 expression and elevated vascular permeability in the lungs. AT effectively inhibited HRPII-mediated neutrophil infiltration, NET formation, and endothelial cell activation in vivo. AT also inhibited HRPII-meditated deposition of platelets and fibrin(ogen) in the lungs and circulating level of von Willebrand factor in the plasma. We conclude that AT exerts protective effects against pathogenic effects of P falciparum-derived HRPII in both cellular and animal models.

A multicenter analytical performance evaluation of a multiplexed immunoarray for the simultaneous measurement of biomarkers of micronutrient deficiency, inflammation and malarial antigenemia.

A lack of comparative data across laboratories is often a barrier to the uptake and adoption of new technologies. Furthermore, data generated by different immunoassay methods may be incomparable due to a lack of harmonization. In this multicenter study, we describe validation experiments conducted in a single lab and cross-lab comparisons of assay results to assess the performance characteristics of the Q-plex™ 7-plex Human Micronutrient Array (7-plex), an immunoassay that simultaneously quantifies seven biomarkers associated with micronutrient (MN) deficiencies, inflammation and malarial antigenemia using plasma or serum; alpha-1-acid glycoprotein, C-reactive protein, ferritin, histidine-rich protein 2, retinol binding protein 4, soluble transferrin receptor, and thyroglobulin. Validations included repeated testing (n = 20 separately prepared experiments on 10 assay plates) in a single lab to assess precision and linearity. Seven independent laboratories tested 76 identical heparin plasma samples collected from a cohort of pregnant women in Niger using the same 7-plex assay to assess differences in results across laboratories. In the analytical validation experiments, intra- and inter-assay coefficients of variation were acceptable at <6% and <15% respectively and assay linearity was 96% to 99% with the exception of ferritin, which had marginal performance in some tests. Cross-laboratory comparisons showed generally good agreement between laboratories in all analyte results for the panel of 76 plasma specimens, with Lin's concordance correlation coefficient values averaging ≥0.8 for all analytes. Excluding plates that would fail routine quality control (QC) standards, the inter-assay variation was acceptable for all analytes except sTfR, which had an average inter-assay coefficient of variation of ≥20%. This initial cross-laboratory study demonstrates that the 7-plex test protocol can be implemented by users with some experience in immunoassay methods, but familiarity with the multiplexed protocol was not essential.