Anti-merozoite antibodies induce natural killer cell effector function and are associated with immunity against malaria.

Natural killer (NK) cells are potent immune effectors that can be activated via antibody-mediated Fc receptor engagement. Using multiparameter flow cytometry, we found that NK cells degranulate and release IFN-γ upon stimulation with antibody-opsonized Plasmodium falciparum merozoites. Antibody-dependent NK (Ab-NK) activity was largely strain transcending and enhanced invasion inhibition into erythrocytes. Ab-NK was associated with the successful control of parasitemia after experimental malaria challenge in African adults. In an independent cohort study in children, Ab-NK increased with age, was boosted by concurrent P. falciparum infections, and was associated with a lower risk of clinical episodes of malaria. Nine of the 14 vaccine candidates tested induced Ab-NK, including some less well-characterized antigens: P41, P113, MSP11, RHOPH3, and Pf_11363200. These data highlight an important role of Ab-NK activity in immunity against malaria and provide a potential mechanism for evaluating vaccine candidates.

Socioeconomic differences in animal food consumption: Education rather than income makes a difference.

Background/aims: Evidence points toward more sustainable and health-conscious dietary behaviors among individuals with higher socioeconomic status. However, these differences vary considerably depending on which indicator of socioeconomic status is examined. Here, we present a systematic parallel investigation of multiple indicators of socioeconomic status as predictors of animal food consumption frequency and selected food-related behaviors in Germany. Methods: Data from the German subsample of two large representative European consumer studies (Study 1 n = 1,954; Study 2 n = 2,045) was used. We assessed the associations between the socioeconomic indicators income, current occupation as well as education and consumption frequency of animal foods and selected food-related behaviors in separate ordinal logistic regressions. Results: Individuals with higher educational attainment engaged in more sustainable and health-conscious dietary behaviors, indicated by significant associations between educational attainment and the consumption frequency of animal foods. Low- and middle-income participants consumed processed meat more frequently (Study 1 only; medium income: OR 1.5, CI 1.09-2.05, p = 0.012; low income: OR 1.43, CI 1.01-2.05, p = 0.047) and fish less frequently (Study 2 only; medium income: OR 0.76, CI 0.59-0.97, p = 0.026; low income: OR 0.061, CI 0.46-0.82, p < 0.001) than participants with high income. Current occupation did not predict the consumption of animal foods or food-related behaviors. Intake frequency of animal-based foods indicates that most participants exceeded national dietary recommendations for meat and processed meat and remained below recommendations for fish and dairy/eggs intake. Conclusion: Educational attainment appears to be the strongest and most consistent socioeconomic indicator of sustainable dietary choices in Germany based on current large, representative studies. Future efforts should be directed toward education interventions about nutrition and interpretation of food labels to compensate for differences in dietary behavior among groups with different levels of education.

Phagocytosis of Plasmodium falciparum ring-stage parasites predicts protection against malaria.

Ring-infected erythrocytes are the predominant asexual stage in the peripheral circulation but are rarely investigated in the context of acquired immunity against Plasmodium falciparum malaria. Here we compare antibody-dependent phagocytosis of ring-infected parasite cultures in samples from a controlled human malaria infection (CHMI) study (NCT02739763). Protected volunteers did not develop clinical symptoms, maintained parasitaemia below a predefined threshold of 500 parasites/µl and were not treated until the end of the study. Antibody-dependent phagocytosis of both ring-infected and uninfected erythrocytes from parasite cultures was strongly correlated with protection. A surface proteomic analysis revealed the presence of merozoite proteins including erythrocyte binding antigen-175 and -140 on ring-infected and uninfected erythrocytes, providing an additional antibody-mediated protective mechanism for their activity beyond invasion-inhibition. Competition phagocytosis assays support the hypothesis that merozoite antigens are the key mediators of this functional activity. Targeting ring-stage parasites may contribute to the control of parasitaemia and prevention of clinical malaria.

The impact of lockdowns during the COVID-19 pandemic on work-related accidents in Austria in 2020.

BACKGROUND: This study aims to investigate the impact of the lockdowns during the COVID-19 (Corona-Virus-Disease 19) pandemic in Austria on work-related accidents in the year 2020. Apart from the lockdowns, multiple work-related measures were introduced in 2020, such as the new law on short-term work and regulation on accidents during home-office. Their combined effects on work-related accidents are unknown and a secondary parameter of this study. METHODS: Daily data on the number of accepted and rejected cases of work-related accidents from the Allgemeine Unfallversicherungsanstalt were obtained for the years 2019 and 2020. Based on data provided by the World Health Organization and government publications, the beginning and end dates of national hard and soft lockdown periods were derived. From this database, a difference-in-differences regression analysis on the absolute number of daily work-related accidents was conducted. RESULTS: On average 272.3 work-related accidents per day were registered in 2019 and 199.4 in 2020, a statistically significant reduction of 72.9 accidents per day and total decrease of 26,164 less accidents compared to 2019. Both lockdowns had a statistically highly significant effect on work-related accidents: The hard lockdown reduced the average number of daily registered work-related accidents by 40%. The light lockdown phases reduced this number by an average of 51%. Weekends and holidays had the greatest impact on work-related accidents with a reduction of 69% and 73%, respectively. CONCLUSION: Both lockdown qualities during the COVID-19 pandemic in Austria led to a significant reduction in work-related accidents for their duration. These findings merit further investigation with more detailed data on sectors and injury-quality.

BCG Provides Short-Term Protection from Experimental Cerebral Malaria in Mice.

Clinical and experimental evidence suggests that the tuberculosis vaccine BCG offers protection against unrelated pathogens including the malaria parasite. Cerebral malaria (CM) is the most severe complication associated with Plasmodium falciparum infection in humans and is responsible for most of the fatalities attributed to malaria. We investigated whether BCG protected C57BL/6 mice from P. berghei ANKA (PbA)-induced experimental CM (ECM). The majority of PbA-infected mice that were immunized with BCG showed prolonged survival without developing clinical symptoms of ECM. However, this protective effect waned over time and was associated with the recovery of viable BCG from liver and spleen. Intriguingly, BCG-mediated protection from ECM was not associated with a reduction in parasite burden, indicating that BCG immunization did not improve anti-parasite effector mechanisms. Instead, we found a significant reduction in pro-inflammatory mediators and CD8+ T cells in brains of BCG-vaccinated mice. Together these data suggest that brain recruitment of immune cells involved in the pathogenesis of ECM decreased after BCG vaccination. Understanding the mechanisms underlying the protective effects of BCG on PbA-induced ECM can provide a rationale for developing effective adjunctive therapies to reduce the risk of death and brain damage in CM.

Transmission of the malaria parasite requires ferlin for gamete egress from the red blood cell.

Ferlins mediate calcium-dependent vesicular fusion. Although conserved throughout eukaryotic evolution, their function in unicellular organisms including apicomplexan parasites is largely unknown. Here, we define a crucial role for a ferlin-like protein (FLP) in host-to-vector transmission of the rodent malaria parasite Plasmodium berghei. Infection of the mosquito vectors requires the formation of free gametes and their fertilisation in the mosquito midgut. Mature gametes will only emerge upon secretion of factors that stimulate the disruption of the red blood cell membrane and the parasitophorous vacuole membrane. Genetic depletion of FLP in sexual stages leads to a complete life cycle arrest in the mosquito. Although mature gametes form normally, mutants lacking FLP remain trapped in the red blood cell. The egress defect is rescued by detergent-mediated membrane lysis. In agreement with ferlin vesicular localisation, HA-tagged FLP labels intracellular speckles, which relocalise to the cell periphery during gamete maturation. Our data define FLP as a novel critical factor for Plasmodium fertilisation and transmission and suggest an evolutionarily conserved example of ferlin-mediated exocytosis.

A Plasmodium Cross-Stage Antigen Contributes to the Development of Experimental Cerebral Malaria.

Cerebral malaria is a complex neurological syndrome caused by an infection with Plasmodium falciparum parasites and is exclusively attributed to a series of host-parasite interactions at the pathological blood-stage of infection. In contrast, the preceding intra-hepatic phase of replication is generally considered clinically silent and thereby excluded from playing any role in the development of neurological symptoms. In this study, however, we present an antigen PbmaLS_05 that is presented to the host immune system by both pre-erythrocytic and intra-erythrocytic stages and contributes to the development of cerebral malaria in mice. Although deletion of the endogenous PbmaLS_05 prevented the development of experimental cerebral malaria (ECM) in susceptible mice after both sporozoite and infected red blood cell (iRBC) infections, we observed significant differences in contribution of the host immune response between both modes of inoculation. Moreover, PbmaLS_05-specific CD8+ T cells contributed to the development of ECM after sporozoite but not iRBC-infection, suggesting that pre-erythrocytic antigens like PbmaLS_05 can also contribute to the development of cerebral symptoms. Our data thus highlight the importance of the natural route of infection in the study of ECM, with potential implications for vaccine and therapeutic strategies against malaria.

Varying Immunizations With Plasmodium Radiation-Attenuated Sporozoites Alter Tissue-Specific CD8+ T Cell Dynamics.

Whole sporozoite vaccines represent one of the most promising strategies to induce protection against malaria. However, the development of efficient vaccination protocols still remains a major challenge. To understand how the generation of immunity is affected by variations in vaccination dosage and frequency, we systematically analyzed intrasplenic and intrahepatic CD8+ T cell responses following varied immunizations of mice with radiation-attenuated sporozoites. By combining experimental data and mathematical modeling, our analysis indicates a reversing role of spleen and liver in the generation of protective liver-resident CD8+ T cells during priming and booster injections: While the spleen acts as a critical source compartment during priming, the increase in vaccine-induced hepatic T cell levels is likely due to local reactivation in the liver in response to subsequent booster injections. Higher dosing accelerates the efficient generation of liver-resident CD8+ T cells by especially affecting their local reactivation. In addition, we determine the differentiation and migration pathway from splenic precursors toward hepatic memory cells thereby presenting a mechanistic framework for the impact of various vaccination protocols on these dynamics. Thus, our work provides important insights into organ-specific CD8+ T cell dynamics and their role and interplay in the formation of protective immunity against malaria.

Protection from experimental cerebral malaria with a single intravenous or subcutaneous whole-parasite immunization.

Cerebral malaria is a life-threatening complication of Plasmodia infection and a major cause of child mortality in Sub-Saharan Africa. We report that protection from experimental cerebral malaria in the rodent model is obtained by a single intravenous or subcutaneous whole-parasite immunization. Whole-parasite immunization with radiation-attenuated sporozoites was equally protective as immunization with non-attenuated sporozoites under chemoprophylaxis. Both immunization regimens delayed the development of blood-stage parasites, but differences in cellular and humoral immune mechanisms were observed. Single-dose whole-parasite vaccination might serve as a relatively simple and feasible immunization approach to prevent life-threatening cerebral malaria.

KILchip v1.0: A Novel Plasmodium falciparum Merozoite Protein Microarray to Facilitate Malaria Vaccine Candidate Prioritization.

Passive transfer studies in humans clearly demonstrated the protective role of IgG antibodies against malaria. Identifying the precise parasite antigens that mediate immunity is essential for vaccine design, but has proved difficult. Completion of the Plasmodium falciparum genome revealed thousands of potential vaccine candidates, but a significant bottleneck remains in their validation and prioritization for further evaluation in clinical trials. Focusing initially on the Plasmodium falciparum merozoite proteome, we used peer-reviewed publications, multiple proteomic and bioinformatic approaches, to select and prioritize potential immune targets. We expressed 109 P. falciparum recombinant proteins, the majority of which were obtained using a mammalian expression system that has been shown to produce biologically functional extracellular proteins, and used them to create KILchip v1.0: a novel protein microarray to facilitate high-throughput multiplexed antibody detection from individual samples. The microarray assay was highly specific; antibodies against P. falciparum proteins were detected exclusively in sera from malaria-exposed but not malaria-naïve individuals. The intensity of antibody reactivity varied as expected from strong to weak across well-studied antigens such as AMA1 and RH5 (Kruskal-Wallis H test for trend: p < 0.0001). The inter-assay and intra-assay variability was minimal, with reproducible results obtained in re-assays using the same chip over a duration of 3 months. Antibodies quantified using the multiplexed format in KILchip v1.0 were highly correlated with those measured in the gold-standard monoplex ELISA [median (range) Spearman's R of 0.84 (0.65-0.95)]. KILchip v1.0 is a robust, scalable and adaptable protein microarray that has broad applicability to studies of naturally acquired immunity against malaria by providing a standardized tool for the detection of antibody correlates of protection. It will facilitate rapid high-throughput validation and prioritization of potential Plasmodium falciparum merozoite-stage antigens paving the way for urgently needed clinical trials for the next generation of malaria vaccines.

Plasmodium attenuation: connecting the dots between early immune responses and malaria disease severity.

Sterile attenuation of Plasmodium parasites at the liver-stage either by irradiation or genetic modification, or at the blood-stage by chemoprophylaxis, has been shown to induce immune responses that can protect against subsequent wild-type infection. However, following certain interventions, parasite attenuation can be incomplete or non-sterile. Instead parasites are rendered developmentally stunted but still capable of establishing an acute infection. In experiments involving Plasmodium berghei ANKA, a model of experimental cerebral malaria, it has been observed that several forms of attenuated parasites do not induce cerebral pathology. In this perspective we collect evidence from studies on murine malaria in particular, and attempt to "connect the dots" between early immune responses and protection from severe cerebral disease, highlighting potential parallels to human infection.

Proteomic analysis of the Plasmodium male gamete reveals the key role for glycolysis in flagellar motility.

BACKGROUND: Gametogenesis and fertilization play crucial roles in malaria transmission. While male gametes are thought to be amongst the simplest eukaryotic cells and are proven targets of transmission blocking immunity, little is known about their molecular organization. For example, the pathway of energy metabolism that power motility, a feature that facilitates gamete encounter and fertilization, is unknown. METHODS: Plasmodium berghei microgametes were purified and analysed by whole-cell proteomic analysis for the first time. Data are available via ProteomeXchange with identifier PXD001163. RESULTS: 615 proteins were recovered, they included all male gamete proteins described thus far. Amongst them were the 11 enzymes of the glycolytic pathway. The hexose transporter was localized to the gamete plasma membrane and it was shown that microgamete motility can be suppressed effectively by inhibitors of this transporter and of the glycolytic pathway. CONCLUSIONS: This study describes the first whole-cell proteomic analysis of the malaria male gamete. It identifies glycolysis as the likely exclusive source of energy for flagellar beat, and provides new insights in original features of Plasmodium flagellar organization.

Concentration of LAS and boron in the Itter. Comparison of measured data with results obtained by simulation with the GREAT-ER software.

The GREAT-ER software was used to calculate linear alkylbenzene sulphonate [LAS] and boron concentrations in the Itter stream in North Rhine-Westfalia, Germany. The aim was to investigate the predictive strength of this newly developed tool and to compare its results to measured data. Substance-specific input data which were used in this scenario were partly generic (e.g. LAS and sodium perborate tetrahydrate consumption figures for Germany) and partly (site-)specific data (e.g. half-life time for LAS elimination). The comparison with the measured data reveals that the model predictions for LAS and boron are correct at least within a factor of two, only if generic German consumption data is applied. By using refined input data, the accuracy can be increased further.