The association between naturally acquired IgG subclass specific antibodies to the PfRH5 invasion complex and protection from Plasmodium falciparum malaria

Understanding the targets and mechanisms of human immunity to malaria is important for advancing the development of highly efficacious vaccines and serological tools for malaria surveillance. The PfRH5 and PfRipr proteins form a complex on the surface of P. falciparum merozoites that is essential for invasion of erythrocytes and are vaccine candidates. We determined IgG subclass responses to these proteins among malaria-exposed individuals in Papua New Guinea and their association with protection from malaria in a longitudinal cohort of children. Cytophilic subclasses, IgG1 and IgG3, were predominant with limited IgG2 and IgG4, and IgG subclass-specific responses were higher in older children and those with active infection. High IgG3 to PfRH5 and PfRipr were significantly and strongly associated with reduced risk of malaria after adjusting for potential confounding factors, whereas associations for IgG1 responses were generally weaker and not statistically significant. Results further indicated that malaria exposure leads to the co-acquisition of IgG1 and IgG3 to PfRH5 and PfRipr, as well as to other PfRH invasion ligands, PfRH2 and PfRH4. These findings suggest that IgG3 responses to PfRH5 and PfRipr may play a significant role in mediating naturally-acquired immunity and support their potential as vaccine candidates and their use as antibody biomarkers of immunity

Plasmodium falciparum reticulocyte-binding homologues are targets of human inhibitory antibodies and play a role in immune evasion

Introduction: Antibodies targeting the blood-stage of Plasmodium falciparum play a critical role in naturally acquired immunity to malaria by limiting blood-stage parasitemia. One mode of action of antibodies is the direct inhibition of merozoite invasion of erythrocytes through targeting invasion ligands. However, evasion of inhibitory antibodies may be mediated in P. falciparum by switching between various ligand-mediated merozoite invasion pathways. Here, we investigated the potential roles of invasion ligands PfRH1, PfRH2a and PfRH2b in immune evasion through phenotypic variation, and their importance as targets of human invasion-inhibitory antibodies. Methods: Serum samples from malaria-exposed children and adults in Kenya were examined for their ability to inhibit P. falciparum invasion, using parasites with disrupted pfrh1, pfrh2a or pfrh2b genes. Results and Discussion: The loss of PfRH1 and PfRH2b substantially impacted on susceptibility to inhibitory antibodies, suggesting that variation in the use of these ligands contributes to immune evasion. The effect was less prominent with loss of PfRH2a. Differential inhibition of the knockout and parental lines points to PfRH1 and PfRH2b as targets of acquired growth inhibitory antibodies whereas PfRH2a appeared to be a minor target. There was limited relatedness of the inhibitory responses between different isolates or compared to parasites with deletions of erythrocyte-binding antigens. This further suggests that there is a substantial amount of antigenic diversity in invasion pathways to facilitate immune evasion. These findings provide evidence that PfRH1 and PfRH2b are significant targets of inhibitory antibodies and variation in their expression may facilitate immune evasion. Targeting of multiple invasion ligands in vaccine design is likely to be required to achieve potent inhibitory antibodies and protective efficacy against malaria

Identifying Immune Correlates of Protection Against Plasmodium falciparum Through a Novel Approach to Account for Heterogeneity in Malaria Exposure

Background: A main criterion to identify malaria vaccine candidates is the proof that acquired immunity against them is associated with protection from disease. The age of the studied individuals, heterogeneous malaria exposure, and assumption of the maintenance of a baseline immune response can confound these associations. Methods: Immunoglobulin G/immunoglobulin M (IgG/ IgM) levels were measured by Luminex(R) in Mozambican children monitored for clinical malaria from birth until 3 years of age, together with functional antibodies. Studied candidates were pre-erythrocytic and erythrocytic antigens, including EBAs/PfRhs, MSPs, DBLs, and novel antigens merely or not previously studied in malaria-exposed populations. Cox regression models were estimated at 9 and 24 months of age, accounting for heterogeneous malaria exposure or limiting follow-up according to the antibody's decay. Results: Associations of antibody responses with higher clinical malaria risk were avoided when accounting for heterogeneous malaria exposure or when limiting the follow-up time in the analyses. Associations with reduced risk of clinical malaria were found only at 24 months old, but not younger children, for IgG breadth and levels of IgG targeting EBA140III-V, CyRPA, DBL5epsilon and DBL3x, together with C1q-fixation activity by antibodies targeting MSP119. Conclusions: Malaria protection correlates were identified, only in children aged 24 months old when accounting for heterogeneous malaria exposure. These results highlight the relevance of considering age and malaria exposure, as well as the importance of not assuming the maintenance of a baseline immune response throughout the follow-up. Results may be misleading if these factors are not considered

Low Levels of Human Antibodies to Gametocyte-Infected Erythrocytes Contrasts the PfEMP1-Dominant Response to Asexual Stages in P. falciparum Malaria.

Vaccines that target Plasmodium falciparum gametocytes have the potential to reduce malaria transmission and are thus attractive targets for malaria control. However, very little is known about human immune responses to gametocytes present in human hosts. We evaluated naturally-acquired antibodies to gametocyte-infected erythrocytes (gametocyte-IEs) of different developmental stages compared to other asexual parasite stages among naturally-exposed Kenyan residents. We found that acquired antibodies strongly recognized the surface of mature asexual-IEs, but there was limited reactivity to the surface of gametocyte-IEs of different stages. We used genetically-modified P. falciparum with suppressed expression of PfEMP1, the major surface antigen of asexual-stage IEs, to demonstrate that PfEMP1 is a dominant target of antibodies to asexual-IEs, in contrast to gametocyte-IEs. Antibody reactivity to gametocyte-IEs was similar to asexual-IEs lacking PfEMP1. Significant antibody reactivity to the surface of gametocytes was observed when outside of the host erythrocyte, including recognition of the major gametocyte antigen, Pfs230. This indicates that there is a deficiency of acquired antibodies to gametocyte-IEs despite the acquisition of antibodies to gametocyte antigens and asexual IEs. Our findings suggest that the acquisition of substantial immunity to the surface of gametocyte-IEs is limited, which may facilitate immune evasion to enable malaria transmission even in the face of substantial host immunity to malaria. Further studies are needed to understand the basis for the limited acquisition of antibodies to gametocytes and whether vaccine strategies can generate substantial immunity

Antibody Responses to Antigenic Targets of Recent Exposure Are Associated With Low-Density Parasitemia in Controlled Human Plasmodium falciparum Infections.

The majority of malaria infections in low transmission settings remain undetectable by conventional diagnostics. A powerful model to identify antibody responses that allow accurate detection of recent exposure to low-density infections is controlled human malaria infection (CHMI) studies in which healthy volunteers are infected with the Plasmodium parasite. We aimed to evaluate antibody responses in malaria-naïve volunteers exposed to a single CHMI using a custom-made protein microarray. All participants developed a blood-stage infection with peak parasite densities up to 100 parasites/μl in the majority of participants (50/54), while the remaining four participants had peak densities between 100 and 200 parasites/μl. There was a strong correlation between parasite density and antibody responses associated with the most reactive blood-stage targets 1 month after CHMI (Etramp 5, GLURP-R2, MSP4 and MSP1-19; Spearman's ρ = 0.82, p < 0.001). Most volunteers developed antibodies against a potential marker of recent exposure: Etramp 5 (37/45, 82%). Our findings justify validation in endemic populations to define a minimum set of antigens needed to detect exposure to natural low-density infections

Dihydroartemisinin-piperaquine for intermittent preventive treatment of malaria during pregnancy and risk of malaria in early childhood: A randomized controlled trial.

BACKGROUND: Intermittent preventive treatment of malaria in pregnancy (IPTp) with dihydroartemisinin-piperaquine (IPTp-DP) has been shown to reduce the burden of malaria during pregnancy compared to sulfadoxine-pyrimethamine (IPTp-SP). However, limited data exist on how IPTp regimens impact malaria risk during infancy. We conducted a double-blinded randomized controlled trial (RCT) to test the hypothesis that children born to mothers given IPTp-DP would have a lower incidence of malaria during infancy compared to children born to mothers who received IPTp-SP. METHODS AND FINDINGS: We compared malaria metrics among children in Tororo, Uganda, born to women randomized to IPTp-SP given every 8 weeks (SP8w, n = 100), IPTp-DP every 8 weeks (DP8w, n = 44), or IPTp-DP every 4 weeks (DP4w, n = 47). After birth, children were given chemoprevention with DP every 12 weeks from 8 weeks to 2 years of age. The primary outcome was incidence of malaria during the first 2 years of life. Secondary outcomes included time to malaria from birth and time to parasitemia following each dose of DP given during infancy. Results are reported after adjustment for clustering (twin gestation) and potential confounders (maternal age, gravidity, and maternal parasitemia status at enrolment).The study took place between June 2014 and May 2017. Compared to children whose mothers were randomized to IPTp-SP8w (0.24 episodes per person year [PPY]), the incidence of malaria was higher in children born to mothers who received IPTp-DP4w (0.42 episodes PPY, adjusted incidence rate ratio [aIRR] 1.92; 95% CI 1.00-3.65, p = 0.049) and nonsignificantly higher in children born to mothers who received IPT-DP8w (0.30 episodes PPY, aIRR 1.44; 95% CI 0.68-3.05, p = 0.34). However, these associations were modified by infant sex. Female children whose mothers were randomized to IPTp-DP4w had an apparently 4-fold higher incidence of malaria compared to female children whose mothers were randomized to IPTp-SP8w (0.65 versus 0.20 episodes PPY, aIRR 4.39, 95% CI 1.87-10.3, p = 0.001), but no significant association was observed in male children (0.20 versus 0.28 episodes PPY, aIRR 0.66, 95% CI 0.25-1.75, p = 0.42). Nonsignificant increases in malaria incidence were observed among female, but not male, children born to mothers who received DP8w versus SP8w. In exploratory analyses, levels of malaria-specific antibodies in cord blood were similar between IPTp groups and sex. However, female children whose mothers were randomized to IPTp-DP4w had lower mean piperaquine (PQ) levels during infancy compared to female children whose mothers received IPTp-SP8w (coef 0.81, 95% CI 0.65-1.00, p = 0.048) and male children whose mothers received IPTp-DP4w (coef 0.72, 95% CI 0.57-0.91, p = 0.006). There were no significant sex-specific differences in PQ levels among children whose mothers were randomized to IPTp-SP8w or IPTp-DP8w. The main limitations were small sample size and childhood provision of DP every 12 weeks in infancy. CONCLUSIONS: Contrary to our hypothesis, preventing malaria in pregnancy with IPTp-DP in the context of chemoprevention with DP during infancy does not lead to a reduced incidence of malaria in childhood; in this setting, it may be associated with an increased incidence of malaria in females. Future studies are needed to better understand the biological mechanisms of in utero drug exposure on drug metabolism and how this may affect the dosing of antimalarial drugs for treatment and prevention during infancy. TRIAL REGISTRATION: ClinicalTrials.gov number NCT02163447

Large expert-curated database for benchmarking document similarity detection in biomedical literature search

Document recommendation systems for locating relevant literature have mostly relied on methods developed a decade ago. This is largely due to the lack of a large offline gold-standard benchmark of relevant documents that cover a variety of research fields such that newly developed literature search techniques can be compared, improved and translated into practice. To overcome this bottleneck, we have established the RElevant LIterature SearcH consortium consisting of more than 1500 scientists from 84 countries, who have collectively annotated the relevance of over 180 000 PubMed-listed articles with regard to their respective seed (input) article/s. The majority of annotations were contributed by highly experienced, original authors of the seed articles. The collected data cover 76% of all unique PubMed Medical Subject Headings descriptors. No systematic biases were observed across different experience levels, research fields or time spent on annotations. More importantly, annotations of the same document pairs contributed by different scientists were highly concordant. We further show that the three representative baseline methods used to generate recommended articles for evaluation (Okapi Best Matching 25, Term Frequency-Inverse Document Frequency and PubMed Related Articles) had similar overall performances. Additionally, we found that these methods each tend to produce distinct collections of recommended articles, suggesting that a hybrid method may be required to completely capture all relevant articles. The established database server located at https://relishdb.ict.griffith.edu.au is freely available for the downloading of annotation data and the blind testing of new methods. We expect that this benchmark will be useful for stimulating the development of new powerful techniques for title and title/abstract-based search engines for relevant articles in biomedical research.Peer reviewe

Varianten der Leishmania major [Yakimov & Schockov, 1915] Delta clpB-Mutante : Virulenz und Immunantwort im Mausmodell

Leishmanien durchlaufen einen biphasischen Lebenszyklus, in dessen Verlauf die Parasiten vom promastigoten zum amastigoten Stadium differenzieren. Während der Übertragung vom Insektenvektor auf den Säugerwirt erfahren die Leishmanien eine Erhöhung der Umgebungstemperatur, die in einer Induktion der Hitzeschockantwort resultiert. Dabei werden HSP70 und HSP90 bereits in der Promastigote konstitutiv exprimiert, und erfahren vor dieser bereits hohen Hintergrundexpression nur eine leichte und vor allen Dingen transiente Expressionssteigerung (Brandau et al., 1995). Das Leishmania HSP100 dagegen wird in der Promastigote nur in marginalen Mengen exprimiert, und erfährt eine deutliche Expressionssteigerung während der Stadiendifferenzierung, die im Amastigoten-Stadium erhalten bleibt (Hübel et al., 1997). Durch die Deletion der beiden für HSP100 codierenden clpB Allele wurde gezeigt, dass die Expression von HSP100 für die Virulenz von L. major von entscheidender Bedeutung ist, und dass ein Selektionsdruck auf die Expression dieses Proteins im Amastigoten-Stadium besteht (Hübel et al., 1997). Das Ziel dieser Arbeit bestand darin, Einblicke in die Funktion von HSP100 zu erhalten. Im Verlauf einer Routine-Mauspassage wurde eine sogenannte L. major Delta clpB escape-Variante isoliert, die trotz des Verlustes der beiden clpB Allele Infektionen in BALB/c Mäusen verursachte. Die Untersuchung dieser escape-Variante im Rahmen dieser Arbeit sollte zur Identifizierung des Mechanismus führen, anhand dessen der Verlust von HSP100 überbrückt werden konnte. Die Untersuchung dieses komplementierenden oder kompensierenden Mechanismus sollte Aufschluss darüber geben, welche molekulare Funktion HSP100 in Leishmania ausübt. Dazu sollten sowohl der Einfluss der escape-Parasiten bzw. des escape-Mechanismus auf die Immunreaktion des Wirtes (beispielhaft im Tiermodell der BALB/c Maus) als auch die virulenten escape-Parasiten selbst untersucht werden. Für die Hefe Saccharomyces cerevisiae konnte bereits gezeigt werden, dass HSP70 und HSP104 zu einem gewissen Grad die Aufgaben des jeweils anderen Proteins übernehmen können. Da bereits bekannt ist, dass Leishmanien unter Selektionsdruck dazu neigen, relevante Gene zu amplifizieren bzw. die Expression des Genproduktes zu erhöhen, sollte in Proteom-Studien untersucht werden, ob auch in Leishmania der Verlust von HSP100 durch die verstärkte Expression anderer, funktionell homologer Gene überbrückt werden konnte. Eine genetische Komplementationsstudie sollte weiterhin darüber Aufschluss geben, ob das Fehlen von HSP100 durch gezielte Überexpression relevanter Genomabschnitte kompensiert werden kann. Dazu sollte eine Cosmid-Genbank hergestellt und in L. major Delta clpB transfiziert werden, um anschließend in Mausinfektionsexperimenten auf virulente Parasiten zu selektieren. Eine Untersuchung der Virluenz-vermittelnden Cosmide sollte dann zur Identifizierung relevanter offener Leserahmen führen, deren Überexpression den Funktionsverlust von HSP100 aufheben können

Human antibodies fix complement to inhibit Plasmodium falciparum invasion of erythrocytes and are associated with protection against malaria.

Antibodies play major roles in immunity to malaria; however, a limited understanding of mechanisms mediating protection is a major barrier to vaccine development. We have demonstrated that acquired human anti-malarial antibodies promote complement deposition on the merozoite to mediate inhibition of erythrocyte invasion through C1q fixation and activation of the classical complement pathway. Antibody-mediated complement-dependent (Ab-C') inhibition was the predominant invasion-inhibitory activity of human antibodies; most antibodies were non-inhibitory without complement. Inhibitory activity was mediated predominately via C1q fixation, and merozoite surface proteins 1 and 2 were identified as major targets. Complement fixation by antibodies was very strongly associated with protection from both clinical malaria and high-density parasitemia in a prospective longitudinal study of children. Ab-C' inhibitory activity could be induced by human immunization with a candidate merozoite surface-protein vaccine. Our findings demonstrate that human anti-malarial antibodies have evolved to function by fixing complement for potent invasion-inhibitory activity and protective immunity