Plasmodium GPI-anchored micronemal antigen is essential for parasite transmission through the mosquito host.

Plasmodium parasites, the eukaryotic pathogens that cause malaria, feature three distinct invasive forms tailored to the host environment they must navigate and invade for life cycle progression. One conserved feature of these invasive forms is the micronemes, apically oriented secretory organelles involved in egress, motility, adhesion, and invasion. Here we investigate the role of GPI-anchored micronemal antigen (GAMA), which shows a micronemal localization in all zoite forms of the rodent-infecting species Plasmodium berghei. ∆GAMA parasites are severely defective for invasion of the mosquito midgut. Once formed, oocysts develop normally, however, sporozoites are unable to egress and exhibit defective motility. Epitope-tagging of GAMA revealed tight temporal expression late during sporogony and showed that GAMA is shed during sporozoite gliding motility in a similar manner to circumsporozoite protein. Complementation of P. berghei knockout parasites with full-length P. falciparum GAMA partially restored infectivity to mosquitoes, indicating conservation of function across Plasmodium species. A suite of parasites with GAMA expressed under the promoters of CTRP, CAP380, and TRAP, further confirmed the involvement of GAMA in midgut infection, motility, and vertebrate infection. These data show GAMA's involvement in sporozoite motility, egress, and invasion, implicating GAMA as a regulator of microneme function.

Distinct immune responses associated with vaccination status and protection outcomes after malaria challenge.

Understanding immune mechanisms that mediate malaria protection is critical for improving vaccine development. Vaccination with radiation-attenuated Plasmodium falciparum sporozoites (PfRAS) induces high level of sterilizing immunity against malaria and serves as a valuable tool for the study of protective mechanisms. To identify vaccine-induced and protection-associated responses during malarial infection, we performed transcriptome profiling of whole blood and in-depth cellular profiling of PBMCs from volunteers who received either PfRAS or noninfectious mosquito bites, followed by controlled human malaria infection (CHMI) challenge. In-depth single-cell profiling of cell subsets that respond to CHMI in mock-vaccinated individuals showed a predominantly inflammatory transcriptome response. Whole blood transcriptome analysis revealed that gene sets associated with type I and II interferon and NK cell responses were increased in prior to CHMI while T and B cell signatures were decreased as early as one day following CHMI in protected vaccinees. In contrast, non-protected vaccinees and mock-vaccinated individuals exhibited shared transcriptome changes after CHMI characterized by decreased innate cell signatures and inflammatory responses. Additionally, immunophenotyping data showed different induction profiles of vδ2+ γδ T cells, CD56+ CD8+ T effector memory (Tem) cells, and non-classical monocytes between protected vaccinees and individuals developing blood-stage parasitemia, following treatment and resolution of infection. Our data provide key insights in understanding immune mechanistic pathways of PfRAS-induced protection and infective CHMI. We demonstrate that vaccine-induced immune response is heterogenous between protected and non-protected vaccinees and that inducted-malaria protection by PfRAS is associated with early and rapid changes in interferon, NK cell and adaptive immune responses. Trial Registration: ClinicalTrials.gov NCT01994525.

PfSRPK1 Regulates Asexual Blood Stage Schizogony and Is Essential for Male Gamete Formation.

Serine/arginine-rich protein kinases (SRPKs) are cell cycle-regulated serine/threonine protein kinases and are important regulators of splicing factors. In this study, we functionally characterize SRPK1 of the human malaria parasite Plasmodium falciparum. P. falciparum SRPK1 (PfSRPK1) was expressed in asexual blood-stage and sexual-stage gametocytes. Pfsrpk1- parasites formed asexual schizonts that generated far fewer merozoites than wild-type parasites, causing reduced replication rates. Pfsrpk1- parasites also showed a severe defect in the differentiation of male gametes, causing a complete block in parasite transmission to mosquitoes. RNA sequencing (RNA-seq) analysis of wild-type PfNF54 and Pfsrpk1- stage V gametocytes suggested a role for PfSRPK1 in regulating transcript splicing and transcript abundance of genes coding for (i) microtubule/cilium morphogenesis-related proteins, (ii) proteins involved in cyclic nucleotide metabolic processes, (iii) proteins involved in signaling such as PfMAP2, (iv) lipid metabolism enzymes, (v) proteins of osmophilic bodies, and (vi) crystalloid components. Our study reveals an essential role for PfSRPK1 in parasite cell morphogenesis and suggests this kinase as a target to prevent malaria transmission from humans to mosquitoes. IMPORTANCE Plasmodium sexual stages represent a critical bottleneck in the parasite life cycle. Gametocytes taken up in an infectious blood meal by female anopheline mosquito get activated to form gametes and fuse to form short-lived zygotes, which transform into ookinetes to infect mosquitoes. In the present study, we demonstrate that PfSRPK1 is important for merozoite formation and critical for male gametogenesis and is involved in transcript homeostasis for numerous parasite genes. Targeting PfSRPK1 and its downstream pathways may reduce parasite replication and help achieve effective malaria transmission-blocking strategies.

A genetically engineered Plasmodium falciparum parasite vaccine provides protection from controlled human malaria infection.

Genetically engineered live Plasmodium falciparum sporozoites constitute a potential platform for creating consistently attenuated, genetically defined, whole-parasite vaccines against malaria through targeted gene deletions. Such genetically attenuated parasites (GAPs) do not require attenuation by irradiation or concomitant drug treatment. We previously developed a P. falciparum (Pf) GAP with deletions in P52, P36, and SAP1 genes (PfGAP3KO) and demonstrated its safety and immunogenicity in humans. Here, we further assessed safety, tolerability, and immunogenicity of the PfGAP3KO vaccine and tested its efficacy against controlled human malaria infection (CHMI) in malaria-naïve subjects. The vaccine was delivered by three (n = 6) or five (n = 8) immunizations with ~200 PfGAP3KO-infected mosquito bites per immunization. PfGAP3KO was safe and well tolerated with no breakthrough P. falciparum blood stage infections. Vaccine-related adverse events were predominately localized urticaria related to the numerous mosquito bites administered per vaccination. CHMI via bites with mosquitoes carrying fully infectious Pf NF54 parasites was carried out 1 month after the last immunization. Half of the study participants who received either three or five PfGAP3KO immunizations remained P. falciparum blood stage negative, as shown by a lack of detection of Plasmodium 18S rRNA in the blood for 28 days after CHMI. Six protected study participants received a second CHMI 6 months later, and one remained completely protected. Thus, the PfGAP3KO vaccine was safe and immunogenic and was capable of inducing protection against sporozoite infection. These results warrant further evaluation of PfGAP3KO vaccine efficacy in dose-range finding trials with an injectable formulation.

Anti-TRAP/SSP2 monoclonal antibodies can inhibit sporozoite infection and may enhance protection of anti-CSP monoclonal antibodies.

Vaccine-induced sterilizing protection from infection by Plasmodium parasites, the pathogens that cause malaria, will be essential in the fight against malaria as it would prevent both malaria-related disease and transmission. Stopping the relatively small number of parasites injected by the mosquito before they can migrate from the skin to the liver is an attractive means to this goal. Antibody-eliciting vaccines have been used to pursue this objective by targeting the major parasite surface protein present during this stage, the circumsporozoite protein (CSP). While CSP-based vaccines have recently had encouraging success in disease reduction, this was only achieved with extremely high antibody titers and appeared less effective for a complete block of infection (i.e., sterile protection). While such disease reduction is important, these and other results indicate that strategies focusing on CSP alone may not achieve the high levels of sterile protection needed for malaria eradication. Here, we show that monoclonal antibodies (mAbs) recognizing another sporozoite protein, TRAP/SSP2, exhibit a range of inhibitory activity and that these mAbs may augment CSP-based protection despite conferring no sterile protection on their own. Therefore, pursuing a multivalent subunit vaccine immunization is a promising strategy for improving infection-blocking malaria vaccines.

Systems analysis of immune responses to attenuated P. falciparum malaria sporozoite vaccination reveals excessive inflammatory signatures correlating with impaired immunity.

Immunization with radiation-attenuated sporozoites (RAS) can confer sterilizing protection against malaria, although the mechanisms behind this protection are incompletely understood. We performed a systems biology analysis of samples from the Immunization by Mosquito with Radiation Attenuated Sporozoites (IMRAS) trial, which comprised P. falciparum RAS-immunized (PfRAS), malaria-naive participants whose protection from malaria infection was subsequently assessed by controlled human malaria infection (CHMI). Blood samples collected after initial PfRAS immunization were analyzed to compare immune responses between protected and non-protected volunteers leveraging integrative analysis of whole blood RNA-seq, high parameter flow cytometry, and single cell CITEseq of PBMCs. This analysis revealed differences in early innate immune responses indicating divergent paths associated with protection. In particular, elevated levels of inflammatory responses early after the initial immunization were detrimental for the development of protective adaptive immunity. Specifically, non-classical monocytes and early type I interferon responses induced within 1 day of PfRAS vaccination correlated with impaired immunity. Non-protected individuals also showed an increase in Th2 polarized T cell responses whereas we observed a trend towards increased Th1 and T-bet+ CD8 T cell responses in protected individuals. Temporal differences in genes associated with natural killer cells suggest an important role in immune regulation by these cells. These findings give insight into the immune responses that confer protection against malaria and may guide further malaria vaccine development. Trial registration: ClinicalTrials.gov NCT01994525.

Longitudinal immune profiling after radiation-attenuated sporozoite vaccination reveals coordinated immune processes correlated with malaria protection.

Background: Identifying immune processes required for liver-stage sterilizing immunity to malaria remains an open problem. The IMRAS trial comprised 5x immunizations with radiation-attenuated sporozoites resulting in 55% protection from subsequent challenge. Methods: To identify correlates of vaccination and protection, we performed detailed systems immunology longitudinal profiling of the entire trial time course including whole blood transcriptomics, detailed PBMC cell phenotyping and serum antigen array profiling of 11 IMRAS radiation-attenuated sporozoite (RAS) vaccinees at up to 21 timepoints each. Results: RAS vaccination induced serum antibody responses to CSP, TRAP, and AMA1 in all vaccinees. We observed large numbers of differentially expressed genes associated with vaccination response and protection, with distinctly differing transcriptome responses elicited after each immunization. These included inflammatory and proliferative responses, as well as increased abundance of monocyte and DC subsets after each immunization. Increases in Vδ2 γδ; T cells and MAIT cells were observed in response to immunization over the course of study, and CD1c+ CD40+ DC abundance was significantly associated with protection. Interferon responses strongly differed between protected and non-protected individuals with high interferon responses after the 1st immunization, but not the 2nd-5th. Blood transcriptional interferon responses were correlated with abundances of different circulating classical and non-classical monocyte populations. Conclusions: This study has revealed multiple coordinated immunological processes induced by vaccination and associated with protection. Our work represents the most detailed immunological profiling of a RAS vaccine trial performed to date and will guide the design and interpretation of future malaria vaccine trials.

OMIP-064: A 27-Color Flow Cytometry Panel to Detect and Characterize Human NK Cells and Other Innate Lymphoid Cell Subsets, MAIT Cells, and γδ T Cells.

This 27-color flow cytometry panel was developed in order to assess immunological changes over the course of an immunization and challenge regimen in two experimental malaria vaccine trials. The aim of the study was to find correlates of vaccine-induced protection. Several studies have indicated that protection against malaria appears to involve immune responses at various immunological sites, with liver-resident responses playing an essential role. As it is not feasible to monitor the immune responses within the liver in humans, this panel is developed with the aim to thoroughly characterize the immune responses over time in blood in addition to detecting changes that might reflect what happens in other immunological sites like the liver. The focus of this panel is to detect several innate lymphoid cell populations, including NK cells and their activation status. Moreover, unconventional T cells like mucosal associated invariant T cells and γδ T cells are assessed in the panel. © 2020 International Society for Advancement of Cytometry.

Sexually transmitted founder HIV-1 viruses are relatively resistant to Langerhans cell-mediated restriction.

A single HIV-1 variant establishes infection of the host after sexual contact. Identifying the phenotypic characteristics of these Transmitted Founder (T/F) viruses is important to understand the restriction mechanisms during transmission. Langerhans cells (LCs) are the mucosal dendritic cell subset that has been shown to have a protective role in HIV-1 transmission. Immature LCs efficiently capture and degrade HIV-1 via langerin-mediated restriction. Here we have investigated the capacity of T/F HIV-1 strains to infect mucosal Langerhans cells (LCs). Notably, most T/F variants efficiently infected immature LCs derived from skin and vaginal tissue in contrast to chronic HIV-1 laboratory strains. Next we screened a panel of T/F viruses and their matched 6-month consensus sequence viruses. Interestingly most T/F variants infected immature LCs whereas donor-matched 6-month consensus sequence viruses had lost the ability to infect LCs. However, we also identified 6-month consensus sequence viruses that had retained an ability to infect LCs similar to that of the donor-matched T/F virus. Moreover, some T/F viruses and 6-month consensus sequence viruses were unable to infect immature LCs. Further analyses indicated that T/F viruses are less sensitive to langerin-mediated restriction. These data suggest that T/F HIV-1 variants have the ability to infect immature LCs, which will facilitate transmission.

C-Type Lectin Receptors in Antiviral Immunity and Viral Escape.

C-type lectin receptors (CLRs) are important pattern recognition receptors involved in recognition and induction of adaptive immunity to pathogens. Certain CLRs play an important role in viral infections as they efficiently interact with viruses. However, it has become clear that deadly viruses subvert the function of CLRs to escape antiviral immunity and promote infection. In particular, viruses target CLRs to suppress or modulate type I interferons that play a central role in the innate and adaptive defense against viruses. In this review, we discuss the function of CLRs in binding to enveloped viruses like HIV-1 and Dengue virus, and how uptake and signaling cascades have decisive effects on the outcome of infection.

Interplay between HIV-1 innate sensing and restriction in mucosal dendritic cells: balancing defense and viral transmission.

Innate sensing of HIV-1 by dendritic cells (DCs) initiates cell-intrinsic signalling programs that direct virus restriction and antiviral defenses. These responses include the production of type I interferon (IFN) and a large number of IFN-stimulated genes (ISGs) with a broad spectrum of antiviral effector functions. Initial interactions of HIV-1 at the mucosal surfaces with DC-expressed innate immune factors including cGAS, TRIM5α and SAMHD1 are predictive of viraemia, inflammation and disease pathogenesis. Here, we review the molecular basis of HIV-1 sensing in the two major mucosal DC subsets, that is, epithelial Langerhans cells and subepithelial CD11c+ conventional DCs. We discuss the concerted actions of the host restriction factors and innate sensors as well as viral evasion mechanisms in determining HIV-1 susceptibility to infection and directing antiviral adaptive immune responses.

HIV-1 blocks the signaling adaptor MAVS to evade antiviral host defense after sensing of abortive HIV-1 RNA by the host helicase DDX3.

The mechanisms by which human immunodeficiency virus 1 (HIV-1) avoids immune surveillance by dendritic cells (DCs), and thereby prevents protective adaptive immune responses, remain poorly understood. Here we showed that HIV-1 actively arrested antiviral immune responses by DCs, which contributed to efficient HIV-1 replication in infected individuals. We identified the RNA helicase DDX3 as an HIV-1 sensor that bound abortive HIV-1 RNA after HIV-1 infection and induced DC maturation and type I interferon responses via the signaling adaptor MAVS. Notably, HIV-1 recognition by the C-type lectin receptor DC-SIGN activated the mitotic kinase PLK1, which suppressed signaling downstream of MAVS, thereby interfering with intrinsic host defense during HIV-1 infection. Finally, we showed that PLK1-mediated suppression of DDX3-MAVS signaling was a viral strategy that accelerated HIV-1 replication in infected individuals.

Flow Cytometry-Based Bead-Binding Assay for Measuring Receptor Ligand Specificity.

In this chapter we describe a fluorescent bead-binding assay, which is an efficient and feasible method to measure interaction between ligands and receptors on cells. In principle, any ligand can be coated on fluorescent beads either directly or via antibodies. Binding between ligand-coated beads and cells can be measured by flow cytometry, which results in an easily quantifiable readout. Furthermore, it allows measuring of binding by specific cell subsets within a mixed cell population. Overall, this method is a convenient and easily standardized assay for measuring binding.

SAMHD1 degradation enhances active suppression of dendritic cell maturation by HIV-1.

A hallmark of HIV-1 infection is the lack of sterilizing immunity. Dendritic cells (DCs) are crucial in the induction of immunity, and lack of DC activation might underlie the absence of an effective anti-HIV-1 response. We have investigated how HIV-1 infection affects maturation of DCs. Our data show that even though DCs are productively infected by HIV-1, infection does not induce DC maturation. HIV-1 infection actively suppresses DC maturation, as HIV-1 infection inhibited TLR-induced maturation of DCs and thereby decreased the immune stimulatory capacity of DCs. Interfering with SAMHD1 restriction further increased infection of DCs, but did not lead to DC maturation. Notably, higher infection observed with SAMHD1 depletion correlated with a stronger suppression of maturation. Furthermore, blocking reverse transcription rescued TLR-induced maturation. These data strongly indicate that HIV-1 replication does not trigger immune activation in DCs, but that HIV-1 escapes immune surveillance by actively suppressing DC maturation independent of SAMHD1. Elucidation of the mechanism of suppression can lead to promising targets for therapy or vaccine design.

Sociodemographic and substance use characteristics of γ hydroxybutyrate (GHB) dependent inpatients and associations with dependence severity.

BACKGROUND: The number of admissions to addiction treatment centers in the Netherlands for gamma hydroxybutyrate (GHB) dependence is rapidly growing. Until now, treatment seeking GHB users have hardly been studied. This study characterizes inpatients in treatment for GHB dependence in terms of sociodemographics, motives for substance use and reasons for seeking treatment. In addition, variables associated with dependence severity are identified. METHODS: Patients were recruited by their therapists at 4 different addiction treatment centers dispersed throughout the Netherlands. They were asked to fill out the questionnaire, including sociodemographic and clinical characteristics, GHB and other drug use, and a modified version of the Drug Use Disorders Identification Test (DUDIT) to screen for GHB dependence. The associations of relevant variables with dependence severity were determined using multiple regression analysis. RESULTS: A total of 75 inpatients (response rate 90.4%) participated in the study. Most patients were young (mean 26.8 ± 9.1) males (73%) with low education (78%) and not employed (48%). Most of them (75%) had started using GHB the year before treatment admission, 42 (56%) frequently combined GHB with sedatives and 26 (35%) frequently combined GHB with stimulants. Dependence severity was strongly associated with sleep problems and the combined use of GHB and stimulants. CONCLUSION: This study shows that sociodemographic characteristics of GHB inpatients are similar to those of problematic users of other club drugs. Sleep problems and combined use of GHB and stimulants were strongly associated with GHB dependence. Together, these factors might help to better identify people at risk for GHB dependence.