Integrated transcriptomic and proteomic analyses ofP. falciparumgametocytes: molecular insight into sex-specific processes and translational repression

Sexual differentiation of malaria parasites into gametocytes in the vertebrate host and subsequent gamete fertilization in mosquitoes is essential for the spreading of the disease. The molecular processes orchestrating these transitions are far from fully understood. Here, we report the first transcriptome analysis of male and female Plasmodium falciparum gametocytes coupled with a comprehensive proteome analysis. In male gametocytes there is an enrichment of proteins involved in the formation of flagellated gametes; proteins involved in DNA replication, chromatin organization and axoneme formation. On the other hand, female gametocytes are enriched in proteins required for zygote formation and functions after fertilization; protein-, lipid- and energy-metabolism. Integration of transcriptome and proteome data revealed 512 highly expressed maternal transcripts without corresponding protein expression indicating large scale translational repression in P. falciparum female gametocytes for the first time. Despite a high degree of conservation between Plasmodium species, 260 of these 'repressed transcripts' have not been previously described. Moreover, for some of these genes, protein expression is only reported in oocysts and sporozoites indicating that repressed transcripts can be partitioned into short- and long-term storage. Finally, these data sets provide an essential resource for identification of vaccine/drug targets and for further mechanistic studies

Molecular Markers for Sensitive Detection of Plasmodium falciparum Asexual Stage Parasites and their Application in a Malaria Clinical Trial.

Plasmodium falciparum parasite life stages respond differently to antimalarial drugs. Sensitive stage-specific molecular assays may help to examine parasite dynamics at microscopically detectable and submicroscopic parasite densities in epidemiological and clinical studies. In this study, we compared the performance of skeleton-binding protein 1 (SBP1), ring-infected erythrocyte surface antigen, Hyp8, ring-exported protein 1 (REX1), and PHISTb mRNA for detecting ring-stage trophozoite-specific transcripts using quantitative reverse transcriptase polymerase chain reaction. Markers were tested on tightly synchronized in vitro parasites and clinical trial samples alongside established markers of parasite density (18S DNA and rRNA) and gametocyte density (Pfs25 mRNA). SBP1 was the most sensitive marker but showed low-level expression in mature gametocytes. Novel markers REX1 and PHISTb showed lower sensitivity but higher specificity for ring-stage trophozoites. Using in vivo clinical trial samples from gametocyte-negative patients, we observed evidence of persisting trophozoite transcripts for at least 14 days postinitiation of treatment. It is currently not clear if these transcripts represent viable parasites that may have implications for clinical treatment outcome or transmission potential

The spectrum of neutralizing and non-neutralizing anti-FVIII antibodies in a nationwide cohort of 788 persons with hemophilia A

Objectives: Anti-factor VIII (FVIII) antibodies have been reported to exhibit both neutralizing and non-neutralizing characteristics. This is the first study investigating the full spectrum of FVIII-specific antibodies, including non-neutralizing antibodies, very-low titer inhibitors, and inhibitors, in a large nationwide population of persons with hemophilia A of all severities. Methods: All persons with hemophilia A (mild (FVIII &gt; 5–40 IU/dL)/moderate [FVIII 1–5 IU/dL)/severe (FVIII &lt; 1 IU/dL)] with an available plasma sample who participated in the sixth Hemophilia in the Netherlands study between 2018 and 2019 were included. The presence of anti-FVIII antibodies of the immunoglobulin A, M, and G isotypes and IgG subclasses, along with antibody titer levels, were assessed using direct-binding ELISAs. FVIII specificity was assessed using a competition-based ELISA approach. The inhibitor status was determined using the Nijmegen ultra-sensitive Bethesda assay (NusBA) and the Nijmegen Bethesda assay (NBA). Results: In total, 788 persons with hemophilia A (336 (42.6%) mild, 123 (15.6%) moderate, 329 (41.8%) severe hemophilia) were included. The median age was 45 years (IQR 24–60), and the majority (50.9%) had over 150 exposure days to FVIII concentrates. Within our population, 144 (18.3%) individuals had non-neutralizing FVIII-specific antibodies, 10 (1.3%) had very low-titer inhibitors (NusBA positive; NBA negative), and 13 (1.6%) had inhibitors (both NusBA and NBA positive). IgG1 was the most abundant FVIII-specific antibody subclass, and the highest titer levels were found for IgG4. In individuals without a reported history of inhibitor development, no clear differences were observed in antibody patterns between those who were minimally or highly exposed to FVIII concentrates. IgG4 subclass antibodies were only observed in persons with a reported history of FVIII inhibitor or in those with a currently detected (very low-titer) inhibitor. Conclusion: In this cross-sectional study, we identified non-neutralizing antibodies in a relatively large proportion of persons with hemophilia A. In contrast, in our population, consisting of persons highly exposed to FVIII concentrates, (very low-titer) inhibitors were detected only in a small proportion of persons, reflecting a well-tolerized population. Hence, our findings suggest that only a small subpopulation of non-neutralizing FVIII-specific antibodies is associated with clinically relevant inhibitors.</p

Comparative assessment of An. gambiae and An. stephensi mosquitoes to determine transmission-reducing activity of antibodies against P. falciparum sexual stage antigens.

BACKGROUND: With the increasing interest in vaccines to interrupt malaria transmission, there is a demand for harmonization of current methods to assess Plasmodium transmission in laboratory settings. Potential vaccine candidates are currently tested in the standard membrane feeding assay (SMFA) that commonly relies on Anopheles stephensi mosquitoes. Other mosquito species including Anopheles gambiae are the dominant malaria vectors for Plasmodium falciparum in sub-Saharan Africa. METHODS: Using human serum and monoclonal pre-fertilization (anti-Pfs48/45) and post-fertilization (anti-Pfs25) antibodies known to effectively inhibit sporogony, we directly compared SMFA based estimates of transmission-reducing activity (TRA) for An. stephensi and An. gambiae mosquitoes. RESULTS: In the absence of transmission-reducing antibodies, average numbers of oocysts were similar between An. gambiae and An. stephensi. Antibody-mediated TRA was strongly correlated between both mosquito species, and absolute TRA estimates for pre-fertilisation monoclonal antibodies (mAb) showed no significant difference between the two species. TRA estimates for IgG of naturally exposed individuals and partially effective concentrations of anti-Pfs25 mAb were higher for An. stephensi than for An. gambiae. CONCLUSION: Our findings support the use of An. stephensi in the SMFA for target prioritization. As a vaccine moves through product development, better estimates of TRA and transmission-blocking activity (TBA) may need to be obtained in epidemiologically relevant parasite-species combination

The spectrum of neutralizing and non-neutralizing anti-FVIII antibodies in a nationwide cohort of 788 persons with hemophilia A

ObjectivesAnti-factor VIII (FVIII) antibodies have been reported to exhibit both neutralizing and non-neutralizing characteristics. This is the first study investigating the full spectrum of FVIII-specific antibodies, including non-neutralizing antibodies, very-low titer inhibitors, and inhibitors, in a large nationwide population of persons with hemophilia A of all severities.MethodsAll persons with hemophilia A (mild (FVIII &gt; 5–40 IU/dL)/moderate [FVIII 1–5 IU/dL)/severe (FVIII &lt; 1 IU/dL)] with an available plasma sample who participated in the sixth Hemophilia in the Netherlands study between 2018 and 2019 were included. The presence of anti-FVIII antibodies of the immunoglobulin A, M, and G isotypes and IgG subclasses, along with antibody titer levels, were assessed using direct-binding ELISAs. FVIII specificity was assessed using a competition-based ELISA approach. The inhibitor status was determined using the Nijmegen ultra-sensitive Bethesda assay (NusBA) and the Nijmegen Bethesda assay (NBA).ResultsIn total, 788 persons with hemophilia A (336 (42.6%) mild, 123 (15.6%) moderate, 329 (41.8%) severe hemophilia) were included. The median age was 45 years (IQR 24–60), and the majority (50.9%) had over 150 exposure days to FVIII concentrates. Within our population, 144 (18.3%) individuals had non-neutralizing FVIII-specific antibodies, 10 (1.3%) had very low-titer inhibitors (NusBA positive; NBA negative), and 13 (1.6%) had inhibitors (both NusBA and NBA positive). IgG1 was the most abundant FVIII-specific antibody subclass, and the highest titer levels were found for IgG4. In individuals without a reported history of inhibitor development, no clear differences were observed in antibody patterns between those who were minimally or highly exposed to FVIII concentrates. IgG4 subclass antibodies were only observed in persons with a reported history of FVIII inhibitor or in those with a currently detected (very low-titer) inhibitor.ConclusionIn this cross-sectional study, we identified non-neutralizing antibodies in a relatively large proportion of persons with hemophilia A. In contrast, in our population, consisting of persons highly exposed to FVIII concentrates, (very low-titer) inhibitors were detected only in a small proportion of persons, reflecting a well-tolerized population. Hence, our findings suggest that only a small subpopulation of non-neutralizing FVIII-specific antibodies is associated with clinically relevant inhibitors

Naturally acquired immunity to sexual stage P. falciparum parasites.

Gametocytes are the specialized form of Plasmodium parasites that are responsible for human-to-mosquito transmission of malaria. Transmission of gametocytes is highly effective, but represents a biomass bottleneck for the parasite that has stimulated interest in strategies targeting the transmission stages separately from those responsible for clinical disease. Studying targets of naturally acquired immunity against transmission-stage parasites may reveal opportunities for novel transmission reducing interventions, particularly the development of a transmission blocking vaccine (TBV). In this review, we summarize the current knowledge on immunity against the transmission stages of Plasmodium. This includes immune responses against epitopes on the gametocyte-infected erythrocyte surface during gametocyte development, as well as epitopes present upon gametocyte activation in the mosquito midgut. We present an analysis of historical data on transmission reducing immunity (TRI), as analysed in mosquito feeding assays, and its correlation with natural recognition of sexual stage specific proteins Pfs48/45 and Pfs230. Although high antibody titres towards either one of these proteins is associated with TRI, the presence of additional, novel targets is anticipated. In conclusion, the identification of novel gametocyte-specific targets of naturally acquired immunity against different gametocyte stages could aid in the development of potential TBV targets and ultimately an effective transmission blocking approach

Proguanil and cycloguanil are organic cation transporter and multidrug and toxin extrusion substrates

Abstract Background Malaria, HIV/AIDS, and tuberculosis endemic areas show considerable geographical overlap, leading to incidence of co-infections. This requires treatment with multiple drugs, potentially causing adverse drug–drug interactions (DDIs). As anti-malarials are generally positively charged at physiological pH, they are likely to interact with human organic cation transporters 1 and 2 (OCT1 and OCT2). These transporters are involved in the uptake of drugs into hepatocytes and proximal tubule cells for subsequent metabolic conversion or elimination. This efflux of cationic drugs from hepatocytes and proximal tubule cells into bile and urine can be mediated by multidrug and toxin extrusion 1 and 2-K (MATE1 and MATE2-K) transporters, respectively. Methods Here, the interaction of anti-malarials with these transporters was studied in order to predict potential DDIs. Using baculovirus-transduced HEK293 cells transiently expressing human OCT1, OCT2, MATE1 and MATE2K uptake and inhibition was studied by a range of anti-malarials. Results Amodiaquine, proguanil, pyrimethamine and quinine were the most potent inhibitors of 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (ASP) transport, a known substrate of OCT1/2, resulting in half maximal inhibitory concentrations (IC50) of 11, 13, 1.6, and 3.4 µM, respectively. Only quinine had a drug–drug index higher than the cut-off value of 0.1 for OCT2, therefore, in vivo pharmacokinetic studies focusing on DDIs involving this compound and other OCT2-interacting drugs are warranted. Furthermore, proguanil appeared to be a substrate of OCT1 and OCT2 with affinities of 8.1 and 9.0 µM, respectively. Additionally, MATE1 and MATE2-K were identified as putative transport proteins for proguanil. Finally, its metabolite cycloguanil was also identified as an OCT1, OCT2, MATE1 and MATE2-K substrate. Conclusion Anti-malarials can reduce OCT1 and OCT2 transport activity in vitro. Furthermore, proguanil and cycloguanil were found to be substrates of OCT1, OCT2, MATE1 and MATE2-K, highlighting the importance of these transporters in distribution and excretion. As these compounds shares substrate overlap with metformin DDIs can be anticipated during concurrent treatment

Patients with moderate hemophilia A and B with a severe bleeding phenotype have an increased burden of disease

Background: Patients with moderate hemophilia express varying bleeding phenotypes. Objectives: To assess the burden of disease in patients with moderate hemophilia and a mild or severe phenotype incorporating the thrombin generation profile. Methods: This sub-study of the 6th Hemophilia in the Netherlands study, analyzed data of adults with moderate hemophilia A or B. Patient characteristics and information on bleeding tendency, joint status, and quality of life were obtained from electronic patient files and self-reported questionnaires. A severe bleeding phenotype was defined as an annual bleeding rate ≥5, an annual joint bleeding rate ≥3, and/or the use of secondary/tertiary prophylaxis, and a mild phenotype vice versa. TG was measured with the Nijmegen Hemostasis Assay. Results: This study included 116 patients: 21% had a severe phenotype of whom 46% used prophylaxis. Patients with a severe phenotype treated on demand reported a higher median annual bleeding rate (7), annual joint bleeding rate (3), and more frequently an impaired joint (77%) than patients with a severe phenotype on prophylaxis (2; 0; 70%) or patients with a mild phenotype (0; 0; 47%). Furthermore, patients with a severe phenotype treated on demand experienced a more decreased quality of life. Despite similar factor activity levels, patients with a severe phenotype had a lower thrombin peak height and thrombin potential (0.7%; 0.06%) than patients with a mild phenotype (21.3%; 46.8%). Conclusion: Patients with moderate hemophilia and a severe phenotype treated on demand displayed a high burden of disease as well as a low thrombin generation profile advocating them toward more intensive prophylactic treatment.</p